Feed additive composition

ABSTRACT

A method for improving the performance of a subject or for improving digestibility of a raw material in a feed (e.g. nutrient digestibility, such as amino acid digestibility), or for improving nitrogen retention, or for improving dietary phosphorus absorption and retention, or for improving the efficacy of the phytase, or for improving the subject&#39;s resistance to necrotic enteritis or for improving feed conversion ratio (FCR) or for improving weight gain in a subject or for improving feed efficiency in a subject or for modulating (e.g. improving) the immune response of the subject or for reducing populations of pathogenic bacteria in the gastrointestinal tract of a subject, or for reducing nutrient excretion in manure, which method comprising administering to a subject at least one direct fed microbial in combination with a phytase, wherein the phytase is administered to the subject at a dosage of more than about 1500 FTU/kg feed.

CLAIM FOR PRIORITY

This application is claims the benefit of Great Britain Application No.1213801.2, filed on Aug. 3, 2012, all of which is incorporated byreference in its entirety.

FIELD OF INVENTION

The present invention relates to methods for improving feed compositionsusing at least one direct fed microbial in combination with a high dosephytase(s), and to a feed additive composition comprising at least onedirect fed microbial in combination with a high dose phytase(s). Thepresent invention further relates to uses and kits.

BACKGROUND OF THE INVENTION

Necrotic enteritis is an economically important disease in poultryspecies. It arises as a result of a pathogenic Clostridium perfringensinfection, which is often concomitant with coccidiosis infection. Inextreme cases, it results in high levels of mortality and at asub-clinical level results in intestinal damage and detrimental impactson animal performance. Necrotic enteritis in poultry, and negativeeffects thereof on performance has primarily been controlled by the useof in-feed antibiotics. However, the ban on the use of in-feedantibiotics as growth promoters and consumer pressure to reduce the useof antibiotic application to meat production animals, has made it moredifficult for producers to prevent losses arising from C.perfringes andassociated sub-clinical or clinical necrotic enteritis. There istherefore an opportunity to offer solutions which limit or prevent thedamage caused by C. perfringens. One such solution has been to useprobiotics to promote a healthy gut microflora and a stableenterological ecosystem. However further benefits have been observedwith supplementation with enzymes such as proteases and carbohydrases.Although combinations of DFMs with some enzymes have been contemplated,the interaction between DFMs and enzymes has never been fullyunderstood. The present invention relates to novel specific combinationswhich surprisingly significantly improve production performancecharacteristics in animals.

Monogastric animals are known to contain no or negligible amounts ofendogenous phytase in the stomach and small intestine, and are thereforedependent on supplemental plant and/or microbial or fungal phytase forhydrolization of phytic acid in the proximal digestive tract (Pallauf,J. and Rimbach, G. Arch. Anim. Nutr., 1997, Vol.50, pp 301-319).Additional phytase is often added to the feed of monogastric animals,such as poultry and swine feed. Phytate is the major storage form ofphosphorus in cereals and legumes. Through the action of phytase,phytate is generaly hydrolysed to give lower inositol-phosphates andinorganic phosphate.

The present invention seeks to overcome some of the problems associatedwith a reduced beneficial effect of phytase enzymes in animals subjectto clinical or sub-clinical necrotic enteritis.

The present invention further seeks to overcome the problems associatedwith the anti-nutritional properties of phytic acid, particularly inanimals subject to clinical or sub-clinical necrotic enteritis, leadingto improved availability of nutrients, minerals, vitamins and energy andconsequently improved bio-physical characteristics of monogastricanimals

SUMMARY OF INVENTION

A seminal finding of the present invention is that the inventors havefound that there is an unexpected synergistic effect in the combinationof at least one DFM and high levels (>1500 FTU/kg feed) of phytase,which has been shown to improve performance of a subject to a levelgreater than that of a positive control with low levels of C.perfringens and no necrotic enteritis.

The inventors have demonstrated this by considering percentagemortality, body weight gain and FCR for example. These positive effectswere completely unexpected. Without wishing to be bound by theory theinventors believe these affects may be due to a reduced levels of damageto the intestines as a result of pathogen (e.g. Clostridium perfringensand/or Escherichia coli challenge), compared to the control.

The improved performance of animals may be accounted for through theprevention of the establishment and pathology of harmful bacteria in thegut. In addition it is believed that other beneficial effects of theinvention include increased hydrolysis of phytate, which results inincreased ileal digestibility of proteins and minerals. In addition tothe benefit of increasing nutrient availability to the subject, this canalso result in less substrate reaching the hind-gut for fermentation bybacteria, making it harder for the pathogens in the gut to establish.Another benefit of the present invention is the reduced mucin productioncompared to when phytase is not supplemented. As a result there is alower level of endogenous losses and again less substrate forfermentation by gut microflora.

In particular, a seminal finding of the present invention is that atleast one direct fed microbial (DFM) in combination with high levels(>1500 FTU/kg feed) of phytase has significant beneficial effects on theperformance of an animal, including improving one or more of thefollowing: feed conversion ratio (FCR), ability to digest a raw material(e.g. nutrient digestibility, such as amino acid digestibility),nitrogen retention, survival, carcass yield, growth rate, weight gain,feed efficiency, animals resistance to necrotic enteritis, immuneresponse of the subject.

Another surprising effect of the present invention is that it can reducenutrient excretion in manure (e.g. reduce nitrogen and phosphorus)content of a subject's manure.

In a further aspect of the present invention there is provided a method:

-   -   i) for improving the performance of a subject or    -   ii) for improving digestibility of a raw material in a feed        (e.g. nutrient digestibility, such as amino acid digestibility),        or    -   iii) for improving phosphorus (e.g. dietary phosphorus)        absorption and retention, or    -   iv) for improving the efficacy of the phytase or    -   v) for improving nitrogen retention, or    -   vi) for improving the subject's resistance to necrotic enteritis        or    -   vii) for improving feed conversion ratio (FCR) or    -   viii) for improving weight gain in a subject or    -   ix) for improving feed efficiency in a subject or    -   x) for modulating (e.g. improving) the immune response of the        subject or    -   xi) for reducing populations of pathogenic bacteria in the        gastrointestinal tract of a subject, or    -   xii) for reducing nutrient excretion in manure,    -   which method comprising administering to a subject at least one        direct fed microbial in combination with a phytase, wherein the        phytase is administered to the subject at a dosage of more than        about 1500 FTU/kg feed.

Another aspect of the present invention is the use of at least onedirect fed microbial in combination with a phytase, wherein the phytaseis used at a dosage of more than about 1500 FTU/kg feed:

-   -   i) for improving the performance of a subject or    -   ii) for improving digestibility of a raw material in a feed        (e.g. nutrient digestibility, such as amino acid digestibility)        or    -   iii) for improving nitrogen retention) or    -   iv) for improving phosphorus (e.g. dietary phosphorus)        absorption and retention or    -   v) for improving the efficacy of the phytase or    -   vi) for improving the subject's resistance to necrotic enteritis        or    -   vii) for improving feed conversion ratio (FCR) or    -   viii) for improving weight gain in a subject or    -   ix) for improving feed efficiency in a subject or    -   x) for modulating (e.g. improving) the immune response of the        subject or    -   xi) for reducing populations of pathogenic bacteria in a the        gastrointestinal tract of a subject or    -   xii) for reducing nutrient excretion in manure.

The present invention yet further provides a feed additive compositioncomprising at least one direct fed microbial in combination with aphytase, wherein the phytase is present in the feed additivecomposition:

-   -   a. at about 30,000 FTU/g composition or more when dosed in a        feed at at least 50 g/metric ton (MT) of feed,    -   b. at about 20,000 FTU/g composition or more when dosed in a        feed at at least 75 g/metric ton (MT) of feed,    -   c. at about 15,000 FTU/g composition or more when dosed in a        feed at at least 100 g/metric ton (MT) of feed,    -   d. at about 15,000 FTU/g composition or more when dosed in a        feed at at least 100 g/metric ton (MT) of feed,    -   e. at about 10,000 FTU/g composition or more when dosed in a        feed at at least 150 g/metric ton (MT) of feed,    -   f. at about 7,500 FTU/g composition or more when dosed in a feed        at at least 200 g/metric ton (MT) of feed,    -   g. at about 5,000 FTU/g composition or more when dosed in a feed        at at least 300 g/metric ton (MT) of feed,    -   and wherein the direct fed microbial is present in the feed        additive composition in a range from 2.5×10³ CFU DFM: 1 FTU        enzyme to 6.7×10⁶ CFU:1 FTU enzyme.

In a further aspect of the present invention, there is provided a kitcomprising a feed additive composition according to the presentinvention and instructions for administration.

In a further aspect the present invention provides a method of preparinga feed additive composition, comprising admixing at least one direct fedmicrobial with a phytase, such that the dosage of phytase in thecomposition is:

-   -   a. about 30,000 FTU/g composition or more when dosed in a feed        at at least 50 g/metric ton (MT) of feed,    -   b. about 20,000 FTU/g composition or more when dosed in a feed        at at least 75 g/metric ton (MT) of feed,    -   c. about 15,000 FTU/g composition or more when dosed in a feed        at at least 100 g/metric ton (MT) of feed,    -   d. about 15,000 FTU/g composition or more when dosed in a feed        at at least 100 g/metric ton (MT) of feed,    -   e. about 10,000 FTU/g composition or more when dosed in a feed        at at least 150 g/metric ton (MT) of feed,    -   f. about 7,500 FTU/g composition or more when dosed in a feed at        at least 200 g/metric ton (MT) of feed,    -   g. about 5,000 FTU/g composition or more when dosed in a feed at        at least 300 g/metric ton (MT) of feed,    -   and the dosage of the direct fed microbial in the feed additive        composition in a range from 2.5×10³ CFU DFM: 1 FTU enzyme to        6.7×10⁶ CFU:1 FTU enzyme, and (optionally) packaging.

In a further aspect the present invention provides a feed comprising afeed additive composition according to the present invention.

The present invention yet further provides a method of preparing afeedstuff comprising admixing a feed component with a feed additivecomposition according to the present invention.

In another aspect, the present invention provides a premix comprising afeed additive composition comprising at least one direct fed microbialin combination with a phytase, wherein the phytase is present in thepremix at:

-   -   a. about 30,000 FTU/g composition or more when dosed in a feed        at at least 50 g/metric ton (MT) of feed,    -   b. about 20,000 FTU/g composition or more when dosed in a feed        at at least 75 g/metric ton (MT) of feed,    -   c. about 15,000 FTU/g composition or more when dosed in a feed        at at least 100 g/metric ton (MT) of feed,    -   d. about 15,000 FTU/g composition or more when dosed in a feed        at at least 100 g/metric ton (MT) of feed,    -   e. about 10,000 FTU/g composition or more when dosed in a feed        at at least 150 g/metric ton (MT) of feed,    -   f. about 7,500 FTU/g composition or more when dosed in a feed at        at least 200 g/metric ton (MT) of feed,    -   g. about 5,000 FTU/g composition or more when dosed in a feed at        at least 300 g/metric ton (MT) of feed,

and the direct fed microbial is present in the premix in a range from2.5×10³ CFU DFM: 1 FTU enzyme to 6.7×10⁶ CFU:1 FTU enzyme, and at leastone mineral and/or at least one vitamin.

The present invention also provides a feed additive compositionaccording to the present invention or a premix according to the presentinvention or a feed comprising a feed additive composition (or premix)according to the present invention for preventing and/or treatingcoccidiosis and/or necrotic enteritis in a subject.

In a yet further aspect of the present invention there is provided amethod of preventing and/or treating necrotic enteritis and/orcoccidiosis wherein an effective amount of a feed additive compositionaccording to the present invention or a premix according to the presentinvention or a feed comprising a feed additive composition (or premix)according to the present invention is administered to a subject.

In one broad aspect the present invention yet further provides a methodor use:

-   -   i) for improving digestibility of a raw material in a feed (e.g.        nutrient digestibility, such as amino acid digestibility), or    -   ii) for improving the performance of a subject or    -   iii) for improving phosphorus (e.g. dietary phosphorus)        absorption and retention, or    -   iv) for improving the efficacy of the phytase or    -   v) for improving nitrogen retention, or    -   vi) for improving the subject's resistance to necrotic enteritis        or    -   vii) for improving feed conversion ratio (FCR) or    -   viii) for improving weight gain in a subject or    -   ix) for improving feed efficiency in a subject or    -   x) for modulating (e.g. improving) the immune response of the        subject or    -   xi) for reducing populations of pathogenic bacteria in the        gastrointestinal tract of a subject, or    -   xii) for reducing nutrient excretion in manure,

which method comprising administering to a subject a high dosagephosphate together with a compound which reduces the pH in the subjectsgastrointestinal tract (GIT). In this broad aspect, suitably thecompound may be an organic acid, an essential oil or an antibiotic. Theorganic acid may be selected from one or more of the group consistingof: citric acid, fumaric acid, formic acid, propionic acid, lactic acidand benzoic acid. The antibiotic may be selected from one or more of thegroup consisting of: tetracycline, chlortetracycline, oxytetracycline,oleandomycin, spiramycin, virginiamycin, bacitracin zinc,flavophospholipol, avilamycin and avoparcin. The essential oil (or plantextract) may be selected from one or more of the group consisting ofcinnemaldehyde, thymol, carvacrol, capsaicin, euganol and alicin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of supplementation of EnvivaPro and a HighPhytase Dose (3000 FTU/kg) on Body Weight Gain of broilers raised to 42days under a Necrotic Enteritis challenge.

FIG. 2 shows the effect of supplementation of Enviva Pro and a HighPhytase Dose (3000 FTU/kg) on FCR of broilers raised to 42 days under aNecrotic Enteritis challenge.

FIG. 3 shows the effect of Enviva Pro and High Phytase Dose (3000FTU/kg) on mortality as a result of a Necrotic Enteritis challenge.

FIG. 4 shows the effect of Enviva Pro and High Phytase Dose (3000FTU/kg) on the severity of intestinal damage due to a Necrotic Enteritischallenge.

FIG. 5 shows the effect of different doses of phytase on FCR of broilersfrom 0 to 21 d in the presence and absence of Enviva Pro, under aNecrotic Enteritis challenge.

FIG. 6 shows the effect of different doses of phytase on FCR of broilersfrom day 6-35.

FIG. 7 shows the effect of different doses of HiPhos™ phytase on FCR ofbroilers from day 0 to 21 with and without Enviva Pro, under a NecroticEnteritis challenge.

FIG. 8 shows the effect of different levels of HiPhos™ phytase with andwithout Enviva Pro on tibia ash of broilers at 21 days, under a NecroticEnteritis challenge.

DETAILED DESCRIPTION OF THE INVENTION

Preferably each of the enzymes used in the present invention areexogenous to the DFM. In other words the enzymes are preferably added toor admixed with the DFM. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this disclosurebelongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY, 20 ED., John Wley and Sons, New York (1994), and Hale & Marham,THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991)provide one of skill with a general dictionary of many of the terms usedin this disclosure.

This disclosure is not limited by the exemplary methods and materialsdisclosed herein, and any methods and materials similar or equivalent tothose described herein can be used in the practice or testing ofembodiments of this disclosure. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, any nucleic acidsequences are written left to right in 5′ to 3′ orientation; amino acidsequences are written left to right in amino to carboxy orientation,respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of this disclosure which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

Amino acids are referred to herein using the name of the amino acid, thethree letter abbreviation or the single letter abbreviation.

The term “protein”, as used herein, includes proteins, polypeptides, andpeptides.

As used herein, the term “amino acid sequence” is synonymous with theterm “polypeptide” and/or the term “protein”. In some instances, theterm “amino acid sequence” is synonymous with the term “peptide”. Insome instances, the term “amino acid sequence” is synonymous with theterm “enzyme”.

The terms “protein” and “polypeptide” are used interchangeably herein.In the present disclosure and claims, the conventional one-letter andthree-letter codes for amino acid residues may be used. The 3-lettercode for amino acids as defined in conformity with the IUPACIUB JointCommission on Biochemical Nomenclature (JCBN). It is also understoodthat a polypeptide may be coded for by more than one nucleotide sequencedue to the degeneracy of the genetic code.

Other definitions of terms may appear throughout the specification.Before the exemplary embodiments are described in more detail, it is tounderstand that this disclosure is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present disclosure will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin this disclosure. The upper and lower limits of these smallerranges may independently be included or excluded in the range, and eachrange where either, neither or both limits are included in the smallerranges is also encompassed within this disclosure, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anenzyme” includes a plurality of such candidate agents and reference to“the feed” includes reference to one or more feeds and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that such publicationsconstitute prior art to the claims appended hereto.

The enzymes for use in the present invention can be produced either bysolid or submerged culture, including batch, fed-batch andcontinuous-flow processes. Culturing is accomplished in a growth mediumcomprising an aqueous mineral salts medium, organic growth factors, thecarbon and energy source material, molecular oxygen, and, of course, astarting inoculum of one or more particular microorganism species to beemployed.

The direct fed microbial (DFM) for use in the present invention may bean antipathogen direct fed microbial. The term “antipathogen DFM” asused herein means a DFM that inhibits (e.g. decreases or prevents)intestinal establishment of pathogenic microorganism (such asClostridium perfringens and/or E. coli and/or Salmonella spp and/orCampylobacter spp., preferably Clostridium perfringens and/or E. coli).

The “DFM ASSAY” taught hereinbelow may be used to determine whether theDFM is an antipathogen DFM.

In one embodiment the DFM for use in the present invention is selectedas an inhibitory strain (or an antipathogen DFM) when assessed with the“DFM ASSAY” taught herein.

Suitably the DFM for use in the present invention may inhibit one (ormore) of the following pathogens: Clostridium spp, such as Clostridiumperfringens and/or Clostridium difficile, and/or E. coli and/orSalmonella spp and/or Campylobacter spp, preferably at least one (ormore) of the following pathogens: Clostridium spp, such as Clostridiumperfringens and/or Clostridium difficile, and/or E. coli.

In one embodiment the DFM for use in the present invention may inhibitone (or more) of the following pathogens: Clostridium perfringens and/orClostridium difficile and/or E. coli, preferably Clostridium perfringensand/or Clostridium difficile, more preferably Clostridium perfringens.

In one embodiment the subject may be challenged by a pathogen, such asone or more of the following pathogens: Clostridium spp, such asClostridium perfringens and/or Clostridium difficile, and/or E. coliand/or Salmonella spp and/or Campylobacter spp.

In one embodiment the subject may be challenged by Clostridiumperfringens and/or E. coli. The DFM for use in the present invention issuitably a viable bacterium.

The present invention relates to high phytase dosages.

Conventionally phytase is dosed in the region of about 500 FTU/kg feede.g. for broilers and turkeys, and as is conventionally doses in theregion of about 300 FTU/kg feed for laying hens for example.

In contrast in the present invention the phytase is dosed in (or with)the feedstuff at a level of at least 1500 FTU/kg feed, suitably at least2000 FTU/kg feed, suitably at least 3000 FTU/kg feed, suitably at least5000 FTU/kg feed, such as at least 10000 FTU/kg feed.

As will be understood by one skilled in the art, the level of phytase inthe feed additive composition must be sufficient to allow the phytase tobe dosed in the feedstuff at a level of at least 1500 FTU/kg feed,suitably at least 2000 FTU/kg feed, suitably at least 3000 FTU/kg feed,suitably at least 5000 FTU/kg feed, such as at least 10000 FTU/kg feed.

The present inventors have found that significant benefits can beobtained by administering high phytase levels in combination with DFMs.In contrast, increasing the dose of phytase alone causes no significantbenefits (e.g. no significant improvements in feed conversion ratiosetc.). In sharp contradistinction when the dose of phytase is increasedin combination with at least one DFM (particularly at least antipathogenDFM)—then significant and unexpected benefits are observed. This wascompletely unexpected.

Suitably the DFM for use in the present invention is a mixture of atleast two DFMs, suitably at least three DFMs.

In one embodiment the DFM for use in the present invention comprises (orconsists essentially of or consists of) a bacterium from one or more ofthe following genera: Bacillus, Enterococcus, Pediococcus,Saccharomyces, Bifidobacterium, Lactobacillus, Lactococcus, Aspergillusand combinations thereof.

The DFM for use in the present invention may comprise (or consistessentially of or consist of) a bacterium from one or more of thefollowing species: Bacillus subtilis, Bacillus licheniformis, Bacillusamyloliquefaciens, Bacillus cereus, Enterococcus faecium, Pediococcusacidilactici, Saccharomyces cerevisiae, Bifidobacterium animalis sppanimalis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillussalivarius ssp salivarius, Lactobacillus farciminis, Lactococcus lactis,Clostridium butyricum, Aspergillus oryzae and combinations thereof.

The DFM for use in the present invention may comprise (or consistessentially or consist of) one or more of the following strains:Bacillus subtilis BS18 (NRRL B-50633), Bacillus subtilis BS278 (NRRL50634), Bacillus subtilis 4-7d (NRRL B-50505), Bacillus subtilis 3-5 h(NRRL B-50507), Bacillus subtilis AGTP BS3BP5 (NRRL B-50510), Bacillussubtilis BS918 (NRRL B-50508), Bacillus subtilis AGTP BS1013(NRRL-50509), B. subtilis AGTP 944 (NRRL B-50548), Bacillus subtilisAGTP BS442 (NRRL B-50542), B. subtilis AGTP BS1069 (NRRL B-50544), B.subtilis AGTP BS521 (NRRL B-50545), B. subtilis BS2084 (NRRL B-50013),B. subtilis LSSA01 (NRRL B-50104), B. subtilis 827 (NRRL B-50105), B.subtilis 3A-P4 (PTA-6506), Bacillus subtilis 15A-P4 (PTA-6507), B.subtilis 22C-P1 (PTA-6508), B. subtilis BL21 (NRRL B-50134), Bacilluslicheniformis BL21 (NRRL B-50134), Bacillus licheniformis 3-12a (NRRLB-50504), Bacillus licheniformis 4-2a (NRRL B-50506), Bacilluslicheniformis 842 (NRRL B-50516), Propionibacterium acidipropionici P261(NRRL B-50131), Propionibacterium acidipropionici P179 (NRRL B-50133),Propionibacterium acidipropionici P169 (PTA 5271), Propionibacteriumacidipropionici P170 (PTA 5272), Propionibacterium jensenii P63 (NRRLB-30979), Propionibacterium jensenii P195 (NRRL B-50132), Lactococcuslactis ID7 (PTA 6103), Lactococcus lactis JD19 (PTA 6104), Lactobacillusacidophilus A2020 (NRRL B-30977), Lactobacillus acidophilus A4000h (NRRLB-30978), Lactobacillus acidophilus PIBc6 (NRRL B-50103), Lactobacillusbrevis LBR 1000 (NRRL B-30982), Lactobacillus casei LC222 (NRRLB-30983), Lactobacillus johnsonii PLCB6 (NRRL B-50518), Lactobacillussalivarius o246i33w (NRRL B-50102), Lactobacillus brevis AJ25(PTA-6099), Lactobacillus brevis HE17 (PTA-6100), Lactobacillus brevis1E-1 (PTA-6509), Lactobacillus lactis CI15 (PTA-6101), Lactobacilluslactis DJ6 (PTA-6102), Lactobacillus rhamnosus (CNCM 1-3698),Lactobacillus farciminis (CNCM 1-3699), Enterococcus faecium EF141(EN-1) (NRRL B-30981), Enterococcus faecium 2-1d (NRRL B-50519),Pediococcus acidilactici PlJe3 (NRRL B-50101), Pediococcus acidilacticio246e42 (NRRL B-50171) and combinations thereof.

The DFM for use in the present invention may comprise more than more,suitably more than two, suitably at least three Bacillus spp.

The DFM for use in the present invention may comprise (or consistessentially or consist of) one or more of the following strains:Bacillus subtilis strains 3A-P4 (PTA-6506); 15A-P4 (PTA-6507); 22C-P1(PTA-6508); 2084 (NRRL B-500130); LSSA01 (NRRL-B-50104); BS27 (NRRLB-50105); BS 18 (NRRL B-50633); and BS 278 (NRRL B-50634) orcombinations thereof.

In one embodiment the DFM for use in the present invention may comprise(or consist essentially or consist of) one or more of the followingstrains: Bacillus subtilis strains 15A-P4 (PTA-6507); 2084 (NRRLB-500130); LSSA01 (NRRL-B-50104).

In one embodiment the phytase for use in the present invention is a6-phytase or a 3-phytase, preferably a 6-phytase.

In one embodiment the phytase for use in the present invention may beselected from one or more of the group consisting of: an E. coli phytaseor a Buttiauxella phytase or a Citrobacter phytase or a Hafnia phytaseor an Aspergillus phytase or a Penicillium phytase or a Trichodermaphytase or an E. coli phytase or a Hansenula phytase, or combinationthereof.

In one embodiment the phytase for use in the present invention may be6-phytase from one or more of the following: E. coli, Buttiauxella spp.or Citrobacter spp, e.g. Citrobacter braakii, including combinationsthereof.

Suitably the phytase for use in the present invention may be one or morePhyzymeXP™, AxtraPhy™ or Ronozyme HiPhos™ or Quantum™ or Quantum Blue™.

Suitably the phytase may be a mixture or more than one phytase, such asat least 2 or at least 3 phytases.

In one embodiment preferably the phytase for use in the presentinvention is active at low pH (e.g. is active at between pH2 and pH5.5).

In one embodiment preferably the phytase for use in the presentinvention has an optimum pH at low pH (e.g. has an optimum pH in therange of pH2 and pH5.5).

In one embodiment a clinical or subclinical intestinal disease challengeis present in the subject.

In one embodiment the clinical or subclinical intestinal diseasechallenge may be caused by any pathogen or any pathogenic bacteria, suchas Clostridium perfringens or E.coli.

In one embodiment the dosage of the direct fed microbial in the feedadditive composition may be in a range from 3.8×10³ CFU DFM:1 FTU enzymeto 2.0×10⁵ CFU:1 FTU enzyme.

Direct Fed Microbial (DFM)

The term “microbial” herein is used interchangeably with“microorganism”.

Preferably the DFM comprises a viable microorganism. Preferably the DFMcomprises a viable bacterium or a viable yeast or a viable fungi.

Preferably the DFM comprises a viable bacteria.

The term “viable microorganism” means a microorganism which ismetabolically active or able to differentiate.

In one embodiment the DFM may be a spore forming bacterium and hence theterm DFM may be comprised of or contain spores, e.g. bacterial spores.Therefore in one embodiment the term “viable microorganism” as usedherein may include microbial spores, such as endospores or conidia.

In another embodiment the DFM in the feed additive composition accordingto the present invention is not comprised of or does not containmicrobial spores, e.g. endospores or conidia.

The microorganism may be a naturally occurring microorganism or it maybe a transformed microorganism. The microorganism may also be acombination of suitable microorganisms. In some aspects, the DFMaccording to the present invention may be one or more of the following:a bacterium, a yeast, a fungi.

Preferably the DFM according to the present invention is a probioticmicroorganism.

In the present invention, the term direct fed microbial (DFM)encompasses direct fed bacteria, direct fed yeast, direct fed fungi andcombinations thereof.

Preferably the DFM is a direct fed bacterium.

Preferably the DFM is a combination comprising two or more bacteria,e.g. three or more or four or more; or the DFM is a combinationcomprising two or more bacterial strains, e.g. three or more or four ormore.

Preferably the bacterium or bacteria is or are isolated.

Suitably the DFM may comprise a bacterium from one or more of thefollowing genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus,Pediococcus, Enterococcus, Leuconostoc, Carnobacterium,Propionibacterium, Bifidobacterium, Clostridium and Megasphaera andcombinations thereof.

In one embodiment the DFM may be selected from the following Bacillusspp: Bacillus subtilis, Bacillus cereus, Bacillus licheniformis,Bacillus amyloliquefaciens and Bacillus pumilus.

In one embodiment the DFM may be a Bacillus strain.

In one embodiment the DFM may be selected from the group consisting of:

Bacillus subtilis BS18 (NRRL B-50633), Bacillus subtilis BS278 (NRRL50634), Bacillus subtilis 4-7d (NRRL B-50505), Bacillus subtilis 3-5h(NRRL B-50507), Bacillus subtilis AGTP BS3BP5 (NRRL B-50510), Bacillussubtilis BS918 (NRRL B-50508), Bacillus subtilis AGTP BS1013(NRRL-50509), B. subtilis AGTP 944 (NRRL B-50548), Bacillus subtilisAGTP BS442 (NRRL B-50542), B. subtilis AGTP BS1069 (NRRL B-50544), B.subtilis AGTP BS521 (NRRL B-50545), B. subtilis BS2084 (NRRL B-50013),B. subtilis LSSA01 (NRRL B-50104), B. subtilis B27 (NRRL B-50105), B.subtilis 3A-P4 (PTA-6506), Bacillus subtilis 15A-P4 (PTA-6507), B.subtilis 22C-P1 (PTA-6508), B. subtilis BL21 (NRRL B-50134) andcombinations thereof.

For the avoidance of doubt, B. subtilis LSSA01 (NRRL B-50104) issometimes referred to as strain BS8.

In one embodiment the DFM may be selected from the group consisting of:

Bacillus pumilus strain AGTP BS 1068 (NRRL B-50543) or B. pumilus KX11-1(NRRL B-50546) and combinations thereof.

In one embodiment the DFM may be selected from the group consisting of:Bacillus licheniformis BL21 (NRRL B-50134), Bacillus licheniformis 3-12a(NRRL B-50504), Bacillus licheniformis 4-2a (NRRL B-50506) and Bacilluslicheniformis 842 (NRRL B-50516). In one embodiment the DFM may be acombination comprising two or more Bacillus strains. In one embodimentthe DFM may be a combination of two or more the Bacillus subtilisstrains 3A-P4 (PTA-6506); 15A-P4 (PTA-6507); 22C-P1 (PTA-6508); 2084(NRRL B-500130); LSSA01 (NRRL-B-50104); BS27 (NRRL B-50105); BS 18 (NRRLB-50633); and BS 278 (NRRL B-50634).

In a preferred embodiment the DFM may comprise (or consist essentiallyof or consist of) one of the following combinations:

-   -   B. subtilis LSSA01 (NRRL B-50104)+Bacillus subtilis BS18 (NRRL        B-50633)+B. subtilis 22C-P1 (PTA-6508); or    -   B. subtilis LSSA01 (NRRL B-50104)+B. subtilis 3A-P4        (PTA-6506)+B. subtilis BS2084 (NRRL B-50013); or    -   B. subtilis LSSA01 (NRRL B-50104)+B. subtilis 3A-P4        (PTA-6506)+B. subtilis BS2084 (NRRL B-50013)+Bacillus subtilis        15A-P4 (PTA-6507); or    -   B. subtilis 3A-P4 (PTA-6506)+B. subtilis 22C-P1 (PTA-6508)+B.        subtilis BS2084 (NRRL B-50013); or    -   B. subtilis LSSA01 (NRRL B-50104)+Bacillus subtilis 15A-P4        (PTA-6507)+B. subtilis BS2084 (NRRL B-50013); or    -   B. subtilis 3A-P4 (PTA-6506)+Bacillus subtilis BS18 (NRRL        B-50633)+B. subtilis 22C-P1 (PTA-6508); or    -   B. subtilis 3A-P4 (PTA-6506)+B. subtilis BS2084 (NRRL        B-50013)+Bacillus subtilis BS278 (NRRL 50634).

Strains 3A-P4 (PTA-6506), 15A-P4 (PTA-6507) and 22C-P1 (PTA-6508) arepublically available from American Type Culture Collection (ATCC).

Strains 2084 (NRRL B-500130); LSSA01 (NRRL-B-50104); BS27 (NRRL B-50105)are publically available from the Agricultural Research Service CultureCollection (NRRL). Strain Bacillus subtilis LSSA01 is sometimes referredto as B. subtilis 8.

These strains are taught in U.S. Pat. No. 7,754,469 B2.

Danisco USA Inc. of W227 N752 Westmound Dr. Waukesha, Wis. 53186, USAalso deposited under the Budapest Treaty the following deposits with theAgricultural Research Service Culture Collection (NRRL) at 1815 NorthUniversity Street, Peoria, Ill. 61604, United States of America with thedates of the original deposits and accession numbers detailed below:

Bacillus licheniformis 3- NRRL B-50504 13 May 2011 12a Bacilluslicheniformis 4-2a NRRL B-50506 13 May 2011 Bacillus licheniformis NRRLB-50516 20 May 2011 BL842 Bacillus pumilus AGTP NRRL B-50546 5 Aug. 2011KXII-1 Bacillus pumilus AGTP NRRL B-50543 4 Aug. 2011 BS1068 Bacillussubtilis BS18 NRRL B-50633 9 Jan. 2012 Bacillus subtilis BS278 NRRLB-50634 9 Jan. 2012 Bacillus subtilis 4-7 d NRRL B-50505 13 May 2011Bacillus subtilis 3-5 h NRRL B-50507 13 May 2011 Bacillus subtilis AGTPNRRL B-50510 13 May 2011 BS3BP5 Bacillus subtilis AGTP NRRL B-50508 13May 2011 BS918 Bacillus subtilis AGTP NRRL B-50509 13 May 2011 BS1013Bacillus subtilis AGTP 944 NRRL B-50548 11 Aug. 2011 Bacillus subtilisAGTP NRRL B-50542 4 Aug. 2011 BS442 Bacillus subtilis AGTP NRRL B-505444 Aug. 2011 BS1069 Bacillus subtilis AGTP NRRL B-50545 4 Aug. 2011 BS521

Danisco USA Inc. of W227 N752 Westmound Dr. Waukesha, Wis. 53186, USAauthorises DuPont Nutrition Biosciences ApS of Langebrogade 1, PO Box17, DK-1001, Copenhagen K, Denmark to refer to all of these depositedbiological materials in this patent application and have givenunreserved and irrevocable consent to the deposited material being madeavailable to the public.

In some embodiments the DFM may be a combination comprising the Bacillussubtilis strains as detailed in the table below:

B. subtilis Bs Bs 8 Bs 3A- Bs 15A- Bs Bs Bs 22C- strain 2084 (LSSAO1) P4P4 278 18 P1 DFM X X X X Combination X X X comprises X X X X X X X X X XX X X X X X X X X X X

In one embodiment the DFM may be selected from Lactococcus spp.

In one embodiment the DFM may be selected from one or more of thefollowing Lactococcus spp: Lactococcus cremoris and Lactococcus lactisand combinations thereof.

In one embodiment the DFM may be selected from the group consisting ofthe following strains or a combination thereof:

Lactococcus lactis ID7 PTA 6103 Lactococcus lactis JD19 PTA 6104

In one embodiment the DFM may be a Lactobacillus spp.

In one embodiment the DFM may be selected from the followingLactobacillus spp: Lactobacillus buchneri, Lactobacillus acidophilus,Lactobacillus casei, Lactobacillus kefiri, Lactobacillus bifidus,Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus paracasei,Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacilluscurvatus, Lactobacillus bulgaricus, Lactobacillus sakei, Lactobacillusreuteri, Lactobacillus fermentum, Lactobacillus farciminis,Lactobacillus lactis, Lactobacillus delbreuckii, Lactobacillusplantarum, Lactobacillus paraplantarum, Lactobacillus farciminis,Lactobacillus rhamnosus, Lactobacillus crispatus, Lactobacillus gasseri,Lactobacillus johnsonfi and Lactobacillus jensenfi, and combinations ofany thereof.

In one embodiment the DFM may be selected from the group consisting ofthe following strains or a combination thereof:

Lactobacillus acidophilus A2020 NRRL B-30977 Lactobacillus acidophilusA4000h NRRL B-30978 Lactobacillus acidophilus PIBc6 NRRL B-50103Lactobacillus brevis LBR 1000 NRRL B-30982 Lactobacillus casei LC222NRRL B-30983 Lactobacillus johnsonii PLCB6 NRRL B-50518 Lactobacillussalivarius o246i33w NRRL B-50102 Lactobacillus brevis AJ25 PTA-6099Lactobacillus brevis HE17 PTA-6100 Lactobacillus brevis 1E-1 PTA-6509Lactobacillus lactis CI15 PTA-6101 Lactobacillus lactis DJ6 PTA-6102Lactobacillus rhamnosus CNCM I-3698 Lactobacillus farciminis CNCM I-3699

In one embodiment the DFM may be selected from one or more of thefollowing strains: Lactobacillus rhamnosus CNCM-I-3698 and Lactobacillusfarciminis CNCM-I-3699. These strains were deposited at the CollectionNationale de Cultures de Microorganims (CNCM) 25, Rue due Docteur Roux,F75724 Paris Cedex 15, France on 8 Dec. 2006 by Sorbial, Route de Spay72700 Allonnes, France and all right, title and interest in the depositswere subsequently transferred to Danisco France SAS of 20, Rue deBrunel, 75017 Paris, France.

Danisco France SAS has authorised DuPont Nutrition Biosciences ApS ofLangebrogade 1, PO Box 17, DK-1001, Copenhagen K, Denmark to refer tothese deposited biological materials in this patent application and havegiven unreserved and irrevocable consent to the deposited material beingmade available to the public.

In one embodiment the DFM may be the Sorbiflore® product available fromDanisco Animal Nutrition.

In one embodiment the DFM may be from the genus Pediococcus.

In one embodiment the DFM may be from the following Pediococcus species:Pediococcus acidilactici, such as one or more of the following P.acidilactici strains PlJe3 (NRRL B-50101) or o246e42 (NRRL B-50171).

In one embodiment the DFM may be selected from the followingBifidobacteria spp: Bifidobacterium lactis, Bifidobacterium bifidium,Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium breve,Bifidobacterium infantis, Bifidobacterium catenulatum, Bifidobacteriumpseudocatenulatum, Bifidobacterium adolescentis, and Bifidobacteriumangulatum, and combinations of any thereof.

In one embodiment the DFM may be an Enterococcus spp.

In one embodiment the DFM may be from the species Entercoccus faecium.

In one embodiment the DFM may be selected from the group consisting ofEnterococcus faecium EF141 (EN-1) (NRRL B-30981), Enterococcus faecium2-1d (NRRL B-50519) and combinations thereof.

In one embodiment the DFM may be from the genus Proprionbacterium.

In one embodiment the DFM may be selected from the group consisting ofthe following Proprionbacterium species or a combination thereof:Proprionbacterium acidipropionici and Propionibacterium jensenii.

In one embodiment the DFM may selected from the group consisting of thefollowing Proprionbacterium strains or combinations thereof:

Propionibacterium acidipropionici P261 NRRL B-50131 Propionibacteriumacidipropionici P179 NRRL B-50133 Propionibacterium acidipropionici P169PTA 5271 Propionibacterium acidipropionici P170 PTA 5272Propionibacterium jensenii P63 NRRL B-30979 Propionibacterium jenseniiP195 NRRL B-50132

Suitably the DFM may comprise a bacterium from one or more of thefollowing species: Bacillus subtilis, Bacillus licheniformis, Bacillusamyloliquefaciens, Enterococcus faecium, Enterococcus spp, andPediococcus spp, Lactobacillus spp, Bifidobacterium spp, Lactobacillusacidophilus, Pediococsus acidilactici, Lactococcus lactis,Bifidobacterium bifidum, Bacillus subtilis, Propionibacterium thoenii,Lactobacillus farciminis, Lactobacillus rhamnosus, Megasphaera elsdenii,Clostridium butyricum, Bifidobacterium animalis ssp. animalis,Lactobacillus reuteri, Bacillus cereus, Lactobacillus salivarius ssp.Salivarius, Propionibacteria sp and combinations thereof.

The direct fed bacterium used in the present invention may be of thesame type (genus, species and strain) or may comprise a mixture ofgenera, species and/or strains.

Suitably the DFM according to the present invention may be one or moreof the products or the microorganisms contained in those products as inthe Table below:

Symbiotic Product Name Company Microorganism(s) ingredients EnvivaPro ®. Danisco A/S Bacillus subtilis strain 2084 Accession (formerlyknown No. NRRI B-50013, as Avicorr ®) Bacillus subtilis strain LSSAO1Accession No. NRRL B-50104 and Bacillus subtilis strain 15A-P4 ATCCAccession No. PTA-6507 Calsporin ® Calpis - Japan Bacillus subtilisStrain C3102 Clostat ® Kemin Bacillus subtilis Strain PB6 IndustriesInc. Cylactin ® DSM Enterococcus faecium NCIMB 10415 (SF68) Gallipro ® &Chr. Hansen Bacillus subtilis Strain C3102 GalliproMax ® A/SGallipro ®Tect ® Chr. Hansen Bacillus licheniformis A/S Poultry star ®Biomin, Inc Enterococcus and Pediococcus Fructo- oligosaccharidesProtexin ® Protexin Int Lactobacillus, Bifidobacterium and anotherProflora ® Alpharma Inc. Bacillus subtilis strain QST 713 β-Mos β-mannanoligosaccharides and β-glucans Ecobiol ® & Norel S.A. Bacillusamyloliquefaciens CECT-5940 Ecobiol ® Plus Fortiflora ® Enterococcusfaecium SF68 BioPlus2B ® DSM Bacillus subtilis and Bacilluslicheniformis Lactiferm ® Chr. Hansen Lactic acid bacteria 7Enterococcus faecium CSI ® Danisco A/S Bacillus strain Yea-Sacc ®Alltech Saccharomyces cerevisiae Biomin IMB52 ® Biomin Enterococcusfaecium Biomin C5 ® Biomin Pediococcus acidilactici, Enterococcusfaecium, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteriLactobacillus salivarius ssp. salivarius Biacton ® ChemVet Lactobacillusfarciminis Oralin E1707 ® Chevita GmBH Enterococcus faeciumProbios-pioneer Chr Hansen Enterococcus faecium (2 strains) PDFM ®Lactococcus lactis DSM 11037 Sorbiflore ® Danisco Animal Lactobacillusrhamnosus and Nutrition Lactobacillus farciminis Animavit ® KRKABacillus subtilis Bonvital ® Lactosan GmbH Enterococcus faecium LevucellSB Lallemand Saccharomyces cerevisiae 20 ® Levucell SC 0 & LallemandSaccharomyces cerevisiae SC10 ® ME Bactocell Lallemand Pediococcusacidilacti ActiSaf ® Le Saffre Saccharomyces cerevisiae (formerlyBioSaf ®) Actisaf ® SC47 Le Saffre Saccharomyces cerevisiae NCYC Sc47Miya-Gold ® Miyarisan Clostridium butyricum Pharma Fecinor and Norel S.AEnterococcus faecium Fecinor Plus ® InteSwine ® ntegro Gida veSaccharomyces cerevisiae NCYC R- Ticaret AS 625 represented by RMAssociates Ltd BioSprint ® ProSol SpA Saccharomyces cerevisia Provita ®Provita Enterococcus faecium and Lactobacillus rhamnosus PepSoyGen-C ®Regal BV Bacillus subtilis and (Nutraferma) Aspergillus oryzaeToyocerin ® Rubinum Bacillus cereus TOYOCERIN ® Rubinum Bacillus cereusvar. toyoi NCIMB 40112/CNCM I-1012

In one embodiment suitably the DFM may be Enviva Pro®.

Enviva Pro® is commercially available from Danisco A/S and is acombination of Bacillus strain 2084 Accession No. NRRI B-50013, Bacillusstrain LSSAO₁ Accession No. NRRL B-50104 and Bacillus strain 15A-P4 ATCCAccession No. PTA-6507 (as taught in U.S. Pat. No. 7,754,469B—incorporated herein by reference).

In one embodiment the DFM may be Bacillus licheniformis BL21 (NRRLB-50134). AgTech Products, Inc. of W227 N752 Westmound Drive, Waukesha,Wis. 53186, USA deposited under the Budapest Treaty the followingbiological deposit with the Agricultural Research Service CultureCollection (NRRL) with the date of the original deposit and accessionnumber detailed below:

Bacillus licheniformis BL21 NRRL B-50134 15 Apr. 2008 Enterococcusfaecium NRRL B-30981 30 Oct. 2006 EF141 (EN-1) Enterococcus faecium 2-NRRL B-50519 3 Jun. 2011 1d Lactobacillus acidophilus NRRL B-30977 24Oct. 2006 A2020 Lactobacillus acidophilus NRRL B-30978 24 Oct. 2006A4000h Lactobacillus acidophilus NRRL B-50103 18 Jan. 2008 PIBc6Lactobacillus brevis LBR NRRL B-30982 30 Oct. 2006 1000 Lactobacilluscasei LC222 NRRL B-30983 30 Oct. 2006 Lactobacillus johnsonii NRRLB-50518 6 Jun. 2011 PLCB6 Lactobacillus salivarius NRRL B-50102 18 Jan.2008 o246i33w Pediococcus acidilactici NRRL B-50101 18 Jan. 2008 PIJe3Pediococcus acidilactici NRRL B-50171 29 Aug. 2008 o246e42

AgTech Products, Inc has authorised DuPont Nutrition Biosciences ApS ofLangebrogade 1, PO Box 17, DK-1001, Copenhagen K, Denmark to refer toall of these deposited biological material in this patent applicationand has given unreserved and irrevocable consent to the depositedmaterial being made available to the public.

AgTech Products, Inc. of W227 N752 Westmound Drive, Waukesha, Wis.53186, USA also deposited under the Budapest Treaty the followingbiological deposits with the American Type Culture Collection (ATCC),Manassas, Va. 20110-2209, USA (PTA references) or with the AgriculturalResearch Service Culture Collection (NRRL references) with the dates ofthe original deposits and accession numbers detailed below—these strainsare referred to in the patents listed in the table and as such arepublically available strains:

Lactobacillus brevis AJ25 PTA-6099 22 Jun. 2006 U.S. Pat. No. 7,384,628B Lactobacillus brevis HE17 PTA-6100 22 Jun. 2006 U.S. Pat. No.7,384,628 B Lactobacillus brevis 1E-1 PTA-6509 12 Jan. 2005 U.S. Pat.No. 7,354,757 B Lactobacillus lactis CI15 PTA-6101 22 Jun. 2004 U.S.Pat. No. 7,384,628 B Lactobacillus lactis DJ6 PTA-6102 22 Jun. 2004 U.S.Pat. No. 7,384,628 B Lactococcus lactis ID7 PTA 6103 22 Jun. 2004 U.S.Pat. No. 7,384,628 B Lactococcus lactis JD19 PTA 6104 22 Jun. 2004 U.S.Pat. No. 7,384,628 B Propionibacterium NRRL B- 2 Apr. 2008 U.S. Pat. No.7,470,531 B & acidipropionici P261 50131 U.S. Pat. No. 6,951,643 BPropionibacterium NRRL B- 2 Apr. 2008 U.S. Pat. No. 7,470,531 B &acidipropionici P179 50133 U.S. Pat. No. 6,951,643 B PropionibacteriumPTA 5271 18 Jun. 2003 U.S. Pat. No. 7,470,531 B & acidipropionici P169U.S. Pat. No. 6,951,643 B Propionibacterium PTA 5272 18 Jun. 2003 U.S.Pat. No. 7,470,531 B & acidipropionici P170 U.S. Pat. No. 6,951,643 BPropionibacterium jensenii NRRL B- 24 Oct. 2006 U.S. Pat. No. 5,945,333B P63 30979 Propionibacterium jensenii NRRL B- 2 Apr. 2008 U.S. Pat. No.7,470,531 B & P195 50132 U.S. Pat. No. 6,951,643B

In one embodiment the DFM for use in the present invention is selectedfrom the group consisting of: Bacillus subtilis BS18 (NRRL B-50633),Bacillus subtilis BS278 (NRRL 50634), Bacillus subtilis 4-7d (NRRLB-50505), Bacillus subtilis 3-5h (NRRL B-50507), Bacillus subtilis AGTPBS3BP5 (NRRL B-50510), Bacillus subtilis BS918 (NRRL B-50508), Bacillussubtilis AGTP BS1013 (NRRL-50509), B. subtilis AGTP 944 (NRRL B-50548),Bacillus subtilis AGTP BS442 (NRRL B-50542), B. subtilis AGTP BS1069(NRRL B-50544), B. subtilis AGTP BS521 (NRRL B-50545), B. subtilisBS2084 (NRRL B-50013), B. subtilis LSSA01 (NRRL B-50104), B. subtilis827 (NRRL B-50105), B. subtilis 3A-P4 (PTA-6506), Bacillus subtilis15A-P4 (PTA-6507), B. subtilis 22C-P1 (PTA-6508), B. subtilis BL21 (NRRLB-50134), Bacillus licheniformis BL21 (NRRL B-50134), Bacilluslicheniformis 3-12a (NRRL B-50504), Bacillus licheniformis 4-2a (NRRLB-50506), Bacillus licheniformis 842 (NRRL B-50516), Propionibacteriumacidipropionici P261 (NRRL B-50131), Propionibacterium acidipropioniciP179 (NRRL B-50133), Propionibacterium acidipropionici P169 (PTA 5271),Propionibacterium acidipropionici P170 (PTA 5272), Propionibacteriumjensenii P63 (NRRL B-30979), Propionibacterium jensenii P195 (NRRLB-50132), Lactococcus lactis ID7 (PTA 6103), Lactococcus lactis JD19(PTA 6104), Lactobacillus acidophilus A2020 (NRRL B-30977),Lactobacillus acidophilus A4000h (NRRL B-30978), Lactobacillusacidophilus PlBc6 (NRRL B-50103), Lactobacillus brevis LBR 1000 (NRRLB-30982), Lactobacillus casei LC222 (NRRL B-30983), Lactobacillusjohnsonii PLCB6 (NRRL B-50518), Lactobacillus salivarius o246i33w (NRRLB-50102), Lactobacillus brevis AJ25 (PTA-6099), Lactobacillus brevisHE17 (PTA-6100), Lactobacillus brevis 1E-1 (PTA-6509), Lactobacilluslactis CI15 (PTA-6101), Lactobacillus lactis DJ6 (PTA-6102),Lactobacillus rhamnosus (CNCM 1-3698), Lactobacillus farciminis (CNCM1-3699), Enterococcus faecium EF141 (EN-1) (NRRL B-30981), Enterococcusfaecium 2-1d (NRRL B-50519), Pediococcus acidilactici PlJe3 (NRRLB-50101), Pediococcus acidilactici o246e42 (NRRL B-50171) andcombinations thereof.

Suitably, the DFM may comprise a yeast from the genera: Saccharomycesspp.

Preferably the DFM to be used in accordance with the present inventionis a microorganism which is generally recognised as safe and, which ispreferably GRAS approved.

A skilled person will readily be aware of specific species and orstrains of microorganisms from within the genera described herein whichare used in the food and/or agricultural industries and which aregenerally considered suitable for animal consumption.

Preferably, the DFM used in accordance with the present invention is onewhich is suitable for animal consumption.

Advantageously, where the product is a feed or feed additivecomposition, the viable DFM should remain effective through the normal“sell-by” or “expiration” date of the product during which the feed orfeed additive composition is offered for sale by the retailer. Thedesired lengths of time and normal shelf life will vary from feedstuffto feedstuff and those of ordinary skill in the art will recognise thatshelf-life times will vary upon the type of feedstuff, the size of thefeedstuff, storage temperatures, processing conditions, packagingmaterial and packaging equipment.

In some embodiments it is important that the DFM is tolerant to heat,i.e. is thermotolerant. This is particularly the case where the feed ispelleted. Therefore in one embodiment the DFM may be a thermotolerantmicroorganism, such as a thermotolerant bacteria, including for exampleBacillus spp.

In some embodiments it may be preferable that the DFM is a sporeproducing bacteria, such as Bacilli, e.g. Bacillus spp. Bacilli are ableto from stable endospores when conditions for growth are unfavorable andare very resistant to heat, pH, moisture and disinfectants.

In one embodiment suitably the DFM may decrease or prevent intestinalestablishment of pathogenic microorganism (such as Clostridiumperfringens and/or E. coli and/or Salmonella spp and/or Campylobacterspp., preferably Clostridium perfringens and/or E. coli). The DFMaccording to the present invention may be any suitable DFM. In oneembodiment the following assay “DFM ASSAY” may used to determine thesuitability of a microorganism to be a DFM. For the avoidance of doubtin one embodiment a DFM selected as an inhibitory strain (or anantipathogen DFM) in accordance with the “DFM ASSAY” taught herein is asuitable DFM for use in accordance with the present invention, i.e. inthe feed additive composition according to the present invention.

DFM Assay:

Tubes were seeded each with a representative pathogen from arepresentative cluster. Supernatant from a potential DFM grownaerobically or anaerobically was added to the seeded tubes andincubated.

After incubation, the optical density (OD) of the control andsupernatant treated tubes was measured for each pathogen.

Colonies of (potential DFM) strains that produced a lowered OD comparedwith the control were classified as an inhibitory strain (or anantipathogen DFM).

The DFM assay as used herein is explained in more detail inUS2009/0280090—incorporated herein by reference.

Preferably the representative pathogen used in assay is one (or more) ofthe following: Clostridium, such as Clostridium perfringens and/orClostridium difficile, and/or E. coli and/or Salmonella spp and/orCampylobacter spp. In one preferred embodiment the assay is conductedwith one or more of Clostridium perfringens and/or Clostridium difficileand/or E. coli, preferably Clostridium perfringens and/or Clostridiumdifficile, more preferably Clostridium perfringens.

In one embodiment the DFM of the present invention is preferably anantipathogen.

The term “antipathogen” as used herein means that the DFM counters aneffect (e.g. a negative effect) of a pathogen.

In one embodiment to determine if a DFM is an antipathogen in accordancewith the present invention the above mentioned DFM assay may be used. ADFM is considered to be an antipathogen or an antipathogen DFM if it isclassed as an inhibitory strain in the above mentioned DFM assay,particularly when the pathogen is Clostridium perfringens.

In one embodiment the antipathogen DFM may be one or more of thefollowing bacteria:

Bacillus subtilis strain 2084 Accession No. NRRL B-50013,

Bacillus subtilis strain LSSAO₁ Accession No. NRRL B-50104,0

Bacillus subtilis strain 15A-P4 ATCC Accession No. PTA-6507,

Bacillus subtilis strain 3A-P4 ATCC Accession No. PTA-6506, and

Bacillus subtilis strain BS27 ATCC Accession No. NRRL B-50105.

For the avoidance of doubt these strains are available and are referredto in U.S. Pat. No. 7,754,459 B. In one embodiment the DFM used inaccordance with the present invention is not Lactobacillus gasseri BNR17 Strain Acc No. KCTC 10902BP as taught in WO2008/016214. Preferablythe DFM is not an inactivated microorganism.

In one embodiment the DFM as used here is a composition comprising oneor more DFM microorganisms as described herein. The composition mayadditionally comprise the enzyme of the present invention.

The composition can be fed to an animal as a direct-fed microbial (DFM).

One or more carrier(s) or other ingredients can be added to the DFM.

The DFM may be presented in various physical forms, for example, as atop dress, as a water soluble concentrate for use as a liquid drench orto be added to a milk replacer, gelatin capsule, or gels.

In one embodiment of the top dress form, freeze-dried fermentationproduct is added to a carrier, such as whey, maltodextrin, sucrose,dextrose, limestone (calcium carbonate), rice hulls, yeast culture,dried starch, and/or sodium silico aluminate.

In one embodiment of the water soluble concentrate for a liquid drenchor milk replacer supplement, freeze-dried fermentation product is addedto a water soluble carrier, such as whey, maltodextrin, sucrose,dextrose, dried starch, sodium silico aluminate, and a liquid is addedto form the drench or the supplement is added to milk or a milkreplacer.

In one embodiment of the gelatin capsule form, freeze-dried fermentationproduct is added to a carrier, such as whey, maltodextrin, sugar,limestone (calcium carbonate), rice hulls, yeast culture dried starch,and/or sodium silico aluminate.

In one embodiment, the bacteria and carrier are enclosed in a degradablegelatin capsule. In one embodiment of the gels form, freeze-driedfermentation product is added to a carrier, such as vegetable oil,sucrose, silicon dioxide, polysorbate 80, propylene glycol, butylatedhydroxyanisole, citric acid, ethoxyquin, and/or artificial coloring toform the gel.

The DFM(s) may optionally be admixed with a dry formulation of additivesincluding but not limited to growth substrates, enzymes, sugars,carbohydrates, extracts and growth promoting micro-ingredients. Thesugars could include the following: lactose; maltose; dextrose;malto-dextrin; glucose; fructose; mannose; tagatose; sorbose; raffinose;and galactose. The sugars range from 50-95%, either individually or incombination. The extracts could include yeast or dried yeastfermentation solubles ranging from 5-50%. The growth substrates couldinclude: trypticase, ranging from 5-25%; sodium lactate, ranging from5-30%; and, Tween 80, ranging from 1-5%. The carbohydrates could includemannitol, sorbitol, adonitol and arabitol. The carbohydrates range from5-50% individually or in combination. The micro-ingredients couldinclude the following: calcium carbonate, ranging from 0.5-5.0%; calciumchloride, ranging from 0.5-5.0%; dipotassium phosphate, ranging from0.5-5.0%; calcium phosphate, ranging from 0.5-5.0%; manganeseproteinate, ranging from 0.25-1.00%; and, manganese, ranging from0.25-1.0%.

To prepare DFMs described herein, the culture(s) and carrier(s) (whereused) can be added to a ribbon or paddle mixer and mixed for about 15minutes, although the timing can be increased or decreased. Thecomponents are blended such that a uniform mixture of the cultures andcarriers result. The final product is preferably a dry, flowable powder.The DFM(s) or composition comprising same can then be added to animalfeed or a feed premix, added to an animal's water, or administered inother ways known in the art (preferably simultaneously with the enzymesof the present invention). A feed for an animal can be supplemented withone or more DFM(s) described herein or with a composition describedherein.

By “a mixture of at least two strains,” is meant a mixture of two,three, four, five, six or even more strains. In some embodiments of amixture of strains, the proportions can vary from 1% to 99%. Otherembodiments of a mixture of strains are from 25% to 75%. Additionalembodiments of a mixture of strains are approximately 50% for eachstrain. When a mixture comprises more than two strains, the strains canbe present in substantially equal proportions or in differentproportions in the mixture.

In one embodiment the DFM may be a viable or inviable microorganismwhich is used in isolated or semi-isolated form. The DFM may be used incombination with or without the growth medium in which it was cultured.

In one embodiment, the DFM is capable of producing colony forming unitswhen grown on an appropriate media. The appropriate media may comprise(or consist of) a feed or a feed constituent.

In one embodiment, the DFM is incapable of producing colony formingunits when grown on an appropriate media. The appropriate media maycomprise (or consist of) a feed or a feed constituent.

Irrespective of whether the DFM is capable or incapable of producingcolony forming units when grown on an appropriate media—the cells may bestill metabolically active (e.g. even if they are unable to divide).

In one embodiment the DFM may be administered as inviable cells.

In one embodiment the DFM may be administered as a viable microorganism.

The DFM may be dosed appropriately.

Suitably dosages of DFM in the feed may be between about 1×10³ CFU/gfeed to about 1×10⁹ CFU/g feed, suitably between about 1×10⁴ CFU/g feedto about 1×10⁸ CFU/g feed, suitably between about 7.5×10⁴ CFU/g feed toabout 1×10⁷ CFU/g feed.

In one embodiment the DFM is dosed in the feedstuff at more than about1×10³ CFU/g feed, suitably more than about 1×10⁴ CFU/g feed, suitablymore than about 7.5×10⁴ CFU/g feed.

Suitably dosages of DFM in the feed additive composition may be betweenabout 2×10² CFU/g composition to about 1×10¹⁰ CFU/g composition,suitably between about 1×10³ CFU/g composition to about 1×10¹⁰ CFU/gcomposition, suitably between about 1×10⁴ CFU/g composition to about1×10⁹ CFU/g composition suitably between about 3.75×10⁴ CFU/gcomposition to about 3×10⁸ CFU/g composition.

In one embodiment the DFM is dosed in the feed additive composition atmore than about 1×10⁵ CFU/g composition, suitably more than about 1×10⁶CFU/g composition, suitably more than about 3.75×10⁷ CFU/g composition.

As used herein the term “CFU” means colony forming units and is ameasure of viable cells in which a colony represents an aggregate ofcells derived from a single progenitor cell.

Phytase

As used herein, the term “phytase” refers to an enzyme (i.e. apolypeptide having phytase activity) that catalyzes the hydrolysis ofesters of phosphoric acid, including phytate and phytic acid, andreleases inorganic phosphate.

The phytase for use in the present invention may be classified a6-phytase (classified as E.C. 3.1.3.26) or a 3-phytase (classified asE.C. 3.1.3.8).

In one embodiment the phytase may be a 6-phytase (E.C. 3.1.3.26).

In one preferred embodiment the phytase for use in the present inventionmay be one or more of the phytases in one or more of the commercialproducts below:

Commercial Phytase product ® Company type Phytase source AxtraPHYDanisco Animal 6-phytase Buttiauxella sp. Nutrition Phyzyme XP Danisco6-phytase E. coli gene expressed in Schizosaccahomyces pombe RonozymeDSM/Novozymes 6-phytase Citrobacter braakii gene Hi-Phos expressed inAspergillus (M/L) oryzae Finase ABVista 3-phytase Trichoderma reeseiFinase EC ABVista 6-phytase E. coli gene expressed in Trichoderma reeseiNatuphos BASF 3-phytase Aspergillus Niger Natuzyme Bioproton phytaseTrichoderma (type not longibrachiatum/ specified) Trichoderma reeseiOPTIPHOS ® Huvepharma AD 6-phytase E. coli gene expressed in Pichiapastoris Phytase sp1002 DSM 3-phytase A consensus gene expressed inHansenula polymorpha Quantum Blue ABVista 6-phytase E. coli geneexpressed in Trichoderma Quantum ABVista 6-phytase E. coli geneexpressed in 2500D, Pichia pastoris or 5000L Trichoderma Ronozyme NPDSM/Novozymes 6-phytase Peniphora lycii gene expressed in Aspergillusoryzae Ronozyme P DSM/Novozymes 6-phytase Peniphora lycii gene expressedin Aspergillus oryzae Rovabio PHY Adisseo 3-phytase Penicilliumfuniculosum

The term consensus gene as used herein means that the DNA vector used totransform the organism contains a synthetic phytase gene based on aconsensus sequence, a URA gene from the non-pathogenic yeastSaccharomyces cerevisiae and the origin of replication of theEscherichia coli plasmid pBR322.

In one embodiment the phytase is a Citrobacter phytase derived from e.g.Citrobacter freundii, preferably C. freundii NCIMB 41247 and variantsthereof e.g. as disclosed in WO2006/038062 (incorporated herein byreference) and WO2006/038128 (incorporated herein by reference),Citrobacter braakii YH-15 as disclosed in WO 2004/085638, Citrobacterbraakii ATCC 51113 as disclosed in WO2006/037328 (incorporated herein toreference), as well as variants thereof e.g. as disclosed inWO2007/112739 (incorporated herein by reference) and WO2011/117396(incorporated herein by reference), Citrobacter amalonaticus, preferablyCitrobacter amalonaticus ATCC 25405 or Citrobacter amalonaticus ATCC25407 as disclosed in WO2006037327 (incorporated herein by reference),Citrobacter gillenii, preferably Citrobacter gillenii DSM 13694 asdisclosed in WO2006037327 (incorporated herein by reference), orCitrobacter intermedius, Citrobacter koseri, Citrobacter murliniae,Citrobacter rodentium, Citrobacter sedlakii, Citrobacter werkmanii,Citrobacter youngae, Citrobacter species polypeptides or variantsthereof.

In one embodiment the phytase may be a phytase from Citrobacter, e.g.from Citrobacter freundii, such as the phytase enzyme(s) taught inWO2006/038128, which reference is incorporated herein by reference.

In preferred embodiments, the phytase is preferably E. coli phytasemarketed under the name Phyzyme XP™ by Danisco A/S.

Alternatively the phytase may be a Buttiauxella phytase, e.g. aButtiauxella agrestis phytase, for example, the phytase enzymes taughtin WO 2006/043178, WO 2008/097619, WO2009/129489, WO2006/038128,WO2008/092901, PCT/US2009/41011 or PCT/IB2010/051804, all of which areincorporated herein by reference.

In one aspect, the enzyme used is BP17 or a polypeptide shown in SEQ IDNo. 1 or SEQ ID No. 2 or a variant thereof, such as a sequence having atleast 70% identity thereto, preferably having at least 75% identitythereto, preferably having at least 80% identity thereto, preferablyhaving at least 85% identity thereto, preferably having at least 90%identity thereto, preferably having at least 95% identity thereto,preferably having at least 96% identity thereto, preferably having atleast 97% identity thereto, preferably having at least 98% identitythereto, preferably having at least 99% identity thereto. BP17 is anenzyme variant of a Buttiauxella sp. Phytase and is described in e.g.WO2008/097619, which reference is incorporated herein by reference.

In one embodiment, the enzyme used is BP17 and described in e.g.WO2008/097619. BP17 is an enzyme variant of a Buttiauxella sp. phytase.

The sequence for BP17 (excluding signal peptide) is shown as SEQ IDNo. 1. The sequence for BP17 including the signal peptide (underlines)is shown as SEQ ID No. 2.

SEQ ID NO: 1: NDTPASGYQVEKVVILSRHGVRAPTKMTQTMRDVTPNTWPEWPVKLGYITPRGEHLISLMGGFYRQKFQQQGILSQGSCPTPNSIYVWTDVAQRTLKTGEAFLAGLAPQCGLTIHHQQNLEKADPLFHPVKAGICSMDKTQVQQAVEKEAQTPIDNLNQHYIPSLALMNTTLNFSKSPWCQKHSADKSCDLGLSMPSKLSIKDNGNEVSLDGAIGLSSTLAEIFLLEYAQGMPQAAWGNIHSEQEWALLLKLHNVYFDLMERTPYIARHKGTPLLQAISNALNPNATESKLPDISPDNKILFIAGHDTNIANIAGMLNMRWTLPGQPDNTPPGGALVFERLADKSGKQYVSVSMVYQTLEQLRSQTPLSLNQPAGSVQLKIPGCNDQTAEGYCPLSTFTR VVSQSVEPGCQLQSEQ ID NO: 2:

NDTPASGYQVEKVVILS RHGVRAPTKMTQTMRDVTPNTWPEWPVKLGYITPRGEHLISLMGGFYRQKFQQQGILSQGSCPTPNSIYVWTDVAQRTLKTGEAFLAGLAPQCGLTIHHQQNLEKADPLFHPVKAGICSMDKTQVQQAVEKEAQTPIDNLNQHYIPSLALMNTTLNFSKSPWCQKHSADKSCDLGLSMPSKLSIKDNGNEVSLDGAIGLSSTLAEIFLLEYAQGMPQAAWGNIHSEQEWALLLKLHNVYFDLMERTPYIARHKGTPLLQAISNALNPNATESKLPDISPDNKILFIAGHDTNIANIAGMLNMRWTLPGQPDNTPPGGALVFERLADKSGKQYVSVSMVYQTLEQLRSQTPLSLNQPAGSVQLKIPGCNDQTAEGYCPLSTFTRVVSQSVEPGCQLQ

In one embodiment the phytase is the AxtraPHY™ phytase.

The AxtraPHY™ phytase is also known as the BP17 phytase herein.

In one embodiment the phytase may be a phytase from Hafnia, e.g. fromHafnia alvei, such as the phytase enzyme(s) taught in US2008263688,which reference is incorporated herein by reference.

In one embodiment the phytase may be a phytase from Aspergillus, e.g.from Apergillus orzyae.

In one embodiment the phytase may be a phytase from Penicillium, e.g.from Penicillium funiculosum.

The term “phytase” may be one phytase or a combination of phytasesunless the context clearly dictates otherwise.

Preferably, the phytase is present in the feedstuff in range of about1500 FTU/kg to about 20000 FTU/kg feed, suitably about 1500 FTU/kg toabout 10000 FTU/kg feed, suitably about 1500 FTU/kg feed to about 5000FTU/kg feed, suitably about 1500 FTU/kg feed to about 3000 FTU/kg feed,more preferably about 2000 FTU/kg feed to about 20000 FTU/kg feed,suitably about 2000 FTU/kg feed to about 10000 FTU/kg feed, suitablyabout 2000 FTU/kg feed to about 5000 FTU/kg feed, suitably about 2000FTU/kg feed to about 3500 FTU/kg feed.

In one embodiment the phytase is present in the feedstuff at more thanabout 1500 FTU/kg feed, suitably more than about 2000 FTU/kg feed,suitably more than about 3000 FTU/kg feed.

In one embodiment the phytase is present in the feedstuff at less thanabout 15000 FTU/kg feed, suitably less than about 12000 FTU/kg feed.

In one embodiment, the phytase is present in the feedstuff in range ofabout 1500 FTU/kg to about 5000 FTU/kg feed.

In one embodiment, the phytase is present in the feedstuff in range ofabout 2000 FTU/kg to about 5000 FTU/kg feed.

In a preferred embodiment, the phytase is present in the feedstuff inrange of about 1500 FTU/kg to about 3000 FTU/kg feed.

In a much preferred embodiment, the phytase is present in the feedstuffin range of about 2000 FTU/kg to about 3000 FTU/kg feed.

In one embodiment, the phytase may be present in the feed additivecomposition:

-   -   a. at about 30,000 FTU/g composition or more when the        composition is dosed (e.g. the manufacturer's recommended dose)        in a feed is equal to or greater than 50 g/metric ton (MT) of        feed,    -   b. at about 20,000 FTU/g composition or more when the        composition is dosed (e.g. the manufacturer's recommended dose)        in a feed is equal to or greater than 75 g/metric ton (MT) of        feed,    -   c. at about 15,000 FTU/g composition or more when the        composition is dosed (e.g. the manufacturer's recommended dose)        in a feed is equal to or greater than 100 g/metric ton (MT) of        feed,    -   d. at about 15,000 FTU/g composition or more when the        composition is dosed (e.g. the manufacturer's recommended dose)        in a feed is equal to or greater than 100 g/metric ton (MT) of        feed,    -   e. at about 10,000 FTU/g composition or more when the        composition is dosed (e.g. the manufacturer's recommended dose)        in a feed is equal to or greater than 150 g/metric ton (MT) of        feed,    -   f. at about 7,500 FTU/g composition or more when the composition        is dosed (e.g. the manufacturer's recommended dose) in a feed is        equal to or greater than 200 g/metric ton (MT) of feed,    -   g. at about 5,000 FTU/g composition or more when the composition        is dosed (e.g. the manufacturer's recommended dose) in a feed is        equal to or greater than 300 g/metric ton (MT) of feed.

Suitably when the phytase is present in the feed additive composition inthe dosages given above the direct fed microbial is present in the feedadditive composition in a range from 2.5×10³ CFU DFM: 1 FTU enzyme to6.7×10⁶ CFU:1 FTU enzyme, suitably in a range from 3.8×10³ CFU DFM: 1FTU enzyme to 2.0×10⁵ CFU:1 FTU enzyme.

It will be understood that as used herein 1 FTU (phytase unit) isdefined as the amount of enzyme required to release 1 μmol of inorganicorthophosphate from a substrate in one minute under the reactionconditions defined in the ISO 2009 phytase assay—A standard assay fordetermining phytase activity and 1 FTU can be found at InternationalStandard ISO/DIS 30024: 1-17, 2009.

In one embodiment suitably the enzyme is classified using the E.C.classification above, and the E.C. classification designates an enzymehaving that activity when tested in the assay taught herein fordetermining 1 FTU.

Advantages

The interaction of DFMs with enzymes is complicated and without wishingto be bound by theory, it is very surprising that we can see animprovement in the subject's resistance to necrotic enteritis. Prior tothe present invention the specific combination of DFMs and high phytase(e.g. as taught herein) had not been taught for this specific purpose.

In addition or alternatively in terms of animal performance, e.g. feedconversion ratio, there is a synergistic effect in the combination of aDFM (e.g. antipathogen DFM) with phytase, when the phytase is dosedabove 1500 FTU/kg feed. When phytase is supplemented alone, there is anincrease in performance (i.e. reduction in FCR) at 500 and 1500 FTU/kgfeed and a reduction in performance (i.e. an increase in FCR) when thephytase dose exceeds about 1500 FTU/kg feed. This is particularly truein the conditions of a necrotic enteritis challenge. However, when theDFM is added in combination with phytase above 1500 FTU/kg feed, thereis a consistent numerical increase in performance (i.e. reduction in theFCR) compared to the phytase only treatments. Between the level of 3000FTU/kg feed and 10,000 FTU/kg feed there is a large numerical reductionwhich results in the lowest FCR of all of the treatment groups.Surprisingly the combination of DFM (particularly antipathogen DFM) withphytase at more than 1500 FTU/kg feed results in a synergisticcombination.

An advantage of the present invention is that the combination of DFMs(e.g. antipathogen) DFMs with high dose phytase significantly improvesthe efficacy of the phytase to improve animal performance and/orphosphorus retention in a subject.

A further advantage of the present invention is the combination of DFMs(e.g. antipathogen) DFMs with high dose phytase improves phosphorus(e.g. dietary phosphorus) absorption and retention in a subject. Thiscombinational effect was completely unexpected.

One advantage of the present invention is that the feed additivecomposition according to the present invention can avoid the negativeeffects of necrotic enteritis or can be used for improving the subject'sresistance to necrotic enteritis.

Without wishing to be bound by theory, phytase catalyzes the sequentialhydrolysis of phytate, a principal storage form of phosphorus in cerealsand legumes, to less phosphorylated myo-inositol derivatives withconcomitant release of inorganic phosphate. Hydrolysis of phytate causesa reduction of endogenous losses of amino acids to the intestinal lumen.A reduction of endogenous amino acid losses in the intestine reduces theavailability of nitrogen for bacterial growth, which helps the activityof DFMs on inhibition of C. perfringens and other pathogenic bacteria.

Without wishing to be bound by theory adding phytase enzymes andhydrolysing phytate, can result in an increase in the pH of the smallintestine (duodenum, jejenum and/or ileum). Phytase may also actuallyincrease the amount of bacteria of species E. coli and C. perfringens.This may be associated with a higher pH. The present inventiors havefound that the addition of a DFM (particularly an antipathogen DFM) withhigh doses of phytases may offset the increase in pathogenic bacteria.

High doses of phytase increase the hydrolysis of phytic acid in the gutof animals, which can have advantages for increased digestibility ofphosphorus, calcium, energy, protein and other nutrients and possiblyanimal performance. Surprisingly, however, the inventors have found thathigh doses of phytase cannot achieve the potential improvements onnutritent digestibility and animal performance when clinical orsubclinical intestinal disease challenge is present, e.g. necroticenteritis in broilers, which is a common occurrence in commercial animalproduction. By combining high doses of phytases with an antipathogenicDFM the inventors have surprisingly found that animal performance isfurther enhanced even at very high levels of phytase (e.g. 10,000 FTU/kgfeed).

In combination high dosages of phytase and DFMs surprisingly provides asignificant improvement on the pathogen reduction and/or resistance tonecrotic enteritis and/or feed conversion ratio, and/or body weight gainand/or performance of a subject and/or reducing nutrient excretion inmanure compared with DFMs in combination with standard (low) phytasedosages and/or DFMs alone and/or enzyme (even high dosage phytase)alone.

Formulation of the DFM with the Enzymes

The DFM and the enzyme(s) may be formulated in any suitable way toensure that the formulation comprises viable DFMs and active enzyme(s).

In one embodiment the DFM and enzyme(s) may be formulated as a liquid, adry powder or a granule.

The dry powder or granules may be prepared by means known to thoseskilled in the art, such as, in top-spray fluid bed coater, in a buttomspray Wurster or by drum granulation (e.g.

High sheer granulation), extrusion, pan coating or in a microingredientsmixer.

For some embodiments the DFM and/or the enzyme(s) may be coated, forexample encapsulated. Suitably the DFM and enzyme may be formulatedwithin the same coating or encapsulated within the same capsule.Alternatively the enzyme(s) may be formulated within one coating orencapsulated within one capsule and the DFM could be formulated in acoating separate to the enzyme(s). In some embodiments, such as wherethe DFM is capable of producing endospores, the DFM may be providedwithout any coating. In such circumstances, the DFM endospores may besimply admixed with the enzyme(s). In the latter case, the enzyme(s) maybe coated, e.g. encapsulated.

In one embodiment the coating protects the enzyme(s) from heat and maybe considered a thermoprotectant.

In one embodiment the feed additive composition is formulated to a drypowder or granules as described in WO2007/044968 (referred to as TPTgranules) or WO1997/016076 or WO1992/012645 (each of which isincorporated herein by reference).

In one embodiment the feed additive composition may be formulated to agranule for feed compositions comprising: a core; an active agent; andat least one coating, the active agent of the granule retaining at least50% activity, at least 60% activity, at least 70% activity, at least 80%activity after conditions selected from one or more of a) a feedpelleting process, b) a steam-heated feed pretreatment process, c)storage, d) storage as an ingredient in an unpelleted mixture, and e)storage as an ingredient in a feed base mix or a feed premix comprisingat least one compound selected from trace minerals, organic acids,reducing sugars, vitamins, choline chloride, and compounds which resultin an acidic or a basic feed base mix or feed premix.

With regard to the granule at least one coating may comprise a moisturehydrating material that constitutes at least 55% w/w of the granule;and/or at least one coating may comprise two coatings. The two coatingsmay be a moisture hydrating coating and a moisture barrier coating. Insome embodiments, the moisture hydrating coating may be between 25% and60% w/w of the granule and the moisture barrier coating may be between2% and 15% w/w of the granule. The moisture hydrating coating may beselected from inorganic salts, sucrose, starch, and maltodextrin and themoisture barrier coating may be selected from polymers, gums, whey andstarch.

The granule may be produced using a feed pelleting process and the feedpretreatment process may be conducted between 70° C. and 95° C. for upto several minutes, such as between 85° C. and 95° C.

In one embodiment the feed additive composition may be formulated to agranule for animal feed comprising: a core; an active agent, the activeagent of the granule retaining at least 80% activity after storage andafter a steam-heated pelleting process where the granule is aningredient; a moisture barrier coating; and a moisture hydrating coatingthat is at least 25% w/w of the granule, the granule having a wateractivity of less than 0.5 prior to the steam-heated pelleting process.

The granule may have a moisture barrier coating selected from polymersand gums and the moisture hydrating material may be an inorganic salt.The moisture hydrating coating may be between 25% and 45% w/w of thegranule and the moisture barrier coating may be between 2% and 10% w/wof the granule.

The granule may be produced using a steam-heated pelleting process whichmay be conducted between 85° C. and 95° C. for up to several minutes.

In some embodiments the DFM (e.g. DFM endospores for example) may bediluted using a diluent, such as starch powder, lime stone or the like.

In one embodiment, the composition is in a liquid formulation suitablefor consumption preferably such liquid consumption contains one or moreof the following: a buffer, salt, sorbitol and/or glycerol.

In another embodiment the feed additive composition may be formulated byapplying, e.g. spraying, the enzyme(s) onto a carrier substrate, such asground wheat for example.

In one embodiment the feed additive composition according to the presentinvention may be formulated as a premix. By way of example only thepremix may comprise one or more feed components, such as one or moreminerals and/or one or more vitamins.

In one embodiment the DFM and/or enzyme(s) for use in the presentinvention are formulated with at least one physiologically acceptablecarrier selected from at least one of maltodextrin, limestone (calciumcarbonate), cyclodextrin, wheat or a wheat component, sucrose, starch,Na₂SO₄, Talc, PVA, sorbitol, benzoate, sorbiate, glycerol, sucrose,propylene glycol, 1,3-propane diol, glucose, parabens, sodium chloride,citrate, acetate, phosphate, calcium, etabisulfite, formate and mixturesthereof.

Packaging

In one embodiment the feed additive composition and/or premix and/orfeed or feedstuff according to the present invention is packaged.

In one preferred embodiment the feed additive composition and/or premixand/or feed or feedstuff is packaged in a bag, such as a paper bag.

In an alternative embodiment the feed additive composition and/or premixand/or feed or feedstuff may be sealed in a container. Any suitablecontainer may be used.

Feed or Feedstuff

The feed additive composition of the present invention may be used as—orin the preparation of—a feed.

The term “feed” is used synonymously herein with “feedstuff”.

The feed may be in the form of a solution or as a solid—depending on theuse and/or the mode of application and/or the mode of administration.

When used as—or in the preparation of—a feed—such as functional feed—thecomposition of the present invention may be used in conjunction with oneor more of: a nutritionally acceptable carrier, a nutritionallyacceptable diluent, a nutritionally acceptable excipient, anutritionally acceptable adjuvant, a nutritionally active ingredient.

In a preferred embodiment the feed additive composition of the presentinvention is admixed with a feed component to form a feedstuff.

The term “feed component” as used herein means all or part of thefeedstuff. Part of the feedstuff may mean one constituent of thefeedstuff or more than one constituent of the feedstuff, e.g. 2 or 3 or4. In one embodiment the term “feed component” encompasses a premix orpremix constituents.

Preferably the feed may be a fodder, or a premix thereof, a compoundfeed, or a premix thereof. In one embodiment the feed additivecomposition according to the present invention may be admixed with acompound feed, a compound feed component or to a premix of a compoundfeed or to a fodder, a fodder component, or a premix of a fodder.

The term fodder as used herein means any food which is provided to ananimal (rather than the animal having to forage for it themselves).Fodder encompasses plants that have been cut.

The term fodder includes silage, compressed and pelleted feeds, oils andmixed rations, and also sprouted grains and legumes.

Fodder may be obtained from one or more of the plants selected from:barley rapeseed (canola), corn (maize), millet, oats, sorghum, soybeans,wheat, and legumes.

The term “compound feed” means a commercial feed in the form of a meal,a pellet, nuts, cake or a crumble. Compound feeds may be blended fromvarious raw materials and additives. These blends are formulatedaccording to the specific requirements of the target animal.

Compound feeds can be complete feeds that provide all the daily requirednutrients, concentrates that provide a part of the ration (protein,energy) or supplements that only provide additional micronutrients, suchas minerals and vitamins.

The main ingredients used in compound feed are the feed grains, whichinclude corn, soybeans, sorghum, wheat, oats, and barley.

Suitably a premix as referred to herein may be a composition composed ofmicroingredients such as vitamins, minerals, chemical preservatives,antibiotics, fermentation products, and other essential ingredients.Premixes are usually compositions suitable for blending into commercialrations.

Any feedstuff of the present invention may comprise one or more feedmaterials selected from the group comprising a) cereals, such as smallgrains (e.g., wheat, barley, rye, oats and combinations thereof) and/orlarge grains such as maize or sorghum; b) by products from cereals, suchas corn gluten meal, wet-cake (particularly corn based wet-cake),Distillers Dried Grain (DDG) (particularly corn based Distillers DriedGrain (cDDG)), Distillers Dried Grain Solubles (DDGS) (particularly cornbased Distillers Dried Grain Solubles (cDDGS)), wheat bran, wheatmiddlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel,and citrus pulp; c) protein obtained from sources such as soya,sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal,dried plasma protein, meat and bone meal, potato protein, whey, copra,sesame; d) oils and fats obtained from vegetable and animal sources; e)minerals and vitamins.

A feedstuff of the present invention may contain at least 10%, at least20%, at least 30% or at least 50% by weight corn and soybean meal orcorn and full fat soy, or wheat meal or sunflower meal.

A feedstuff of the present invention may contain between about 0 toabout 40% corn DDGS. If the feedstuff contain any corn DDGS it maycontain between about 5 to about 40% corn DDGS. For poulty—where cornDDGS is present the feedstuff on average may contain between about 7 to15% corn DDGS. For swine (pigs)—where corn DDGS is present the feedstuffmay contain on average 5 to 40% corn DDGS.

A feedstuff of the present invention may contain corn as a single grain,in which case the feedstuff may comprise between about 35% to about 80%corn.

In the present invention the feed may be one or more of the following: acompound feed and premix, including pellets, nuts or (cattle) cake; acrop or crop residue: corn, soybeans, sorghum, oats, barley, copra,chaff, sugar beet waste; fish meal; meat and bone meal; molasses; oilcake and press cake; oligosaccharides; conserved forage plants: silage;seaweed; seeds and grains, either whole or prepared by crushing, millingetc.; sprouted grains and legumes; yeast extract.

The term feed in the present invention also encompasses in someembodiments pet food. A pet food is plant or animal material intendedfor consumption by pets, such as dog food or cat food. Pet food, such asdog and cat food, may be either in a dry form, such as kibble for dogs,or wet canned form. Cat food may contain the amino acid taurine.

The term feed in the present invention also encompasses in someembodiments fish food. A fish food normally contains macro nutrients,trace elements and vitamins necessary to keep captive fish in goodhealth. Fish food may be in the form of a flake, pellet or tablet.Pelleted forms, some of which sink rapidly, are often used for largerfish or bottom feeding species. Some fish foods also contain additives,such as beta carotene or sex hormones, to artificially enhance the colorof ornamental fish.

The term feed in the present invention also encompasses in someembodiment bird food. Bird food includes food that is used both inbirdfeeders and to feed pet birds. Typically bird food comprises of avariety of seeds, but may also encompass suet (beef or mutton fat).

As used herein the term “contacted” refers to the indirect or directapplication of the composition of the present invention to the product(e.g. the feed). Examples of the application methods which may be used,include, but are not limited to, treating the product in a materialcomprising the feed additive composition, direct application by mixingthe feed additive composition with the product, spraying the feedadditive composition onto the product surface or dipping the productinto a preparation of the feed additive composition. In one embodimentthe feed additive composition of the present invention is preferablyadmixed with the product (e.g. feedstuff). Alternatively, the feedadditive composition may be included in the emulsion or raw ingredientsof a feedstuff.

For some applications, it is important that the composition is madeavailable on or to the surface of a product to be affected/treated. Thisallows the composition to impart one or more of the following favourablecharacteristics: performance benefits.

The feed additive compositions of the present invention may be appliedto intersperse, coat and/or impregnate a product (e.g. feedstuff or rawingredients of a feedstuff) with a controlled amount of DFM andenzyme(s).

The DFM and enzyme(s) may be used simultaneously (e.g. when they are inadmixture together or even when they are delivered by different routes)or sequentially (e.g. they may be delivered by different routes). In oneembodiment preferably the DFM and enzyme(s) are applied simultaneously.Preferably the DFM and enzyme(s) are admixed prior to being delivered toa feedstuff or to a raw ingredient of a feedstuff.

The DFM in feed additive compositions according to the presentinvention—can be added in suitable concentrations—such as for example inconcentrations in the final feed product which offer a daily dose ofbetween about 2×10⁵ CFU to about 2×10¹¹ CFU, suitably between about2×10⁶ to about 1×10¹⁰, suitably between about 3.75×10⁷ CFU to about1×10¹⁰ CFU.

Preferably, the feed additive composition of the present invention willbe thermally stable to heat treatment up to about 70° C.; up to about85° C.; or up to about 95° C. The heat treatment may be performed for upto about 1 minute; up to about 5 minutes; up to about 10 minutes; up toabout 30 minutes; up to about 60 minutes. The term thermally stablemeans that at least about 75% of the enzyme components and/or DFM thatwere present/active in the additive before heating to the specifiedtemperature are still present/active after it cools to room temperature.Preferably, at least about 80% of the enzyme components and/or DFM thatwere present and active in the additive before heating to the specifiedtemperature are still present and active after it cools to roomtemperature.

In a particularly preferred embodiment the feed additive composition ishomogenized to produce a powder.

In an alternative preferred embodiment, the feed additive composition isformulated to granules as described in WO2007/044968 (referred to as TPTgranules) incorporated herein by reference.

In another preferred embodiment when the feed additive composition isformulated into granules the granules comprise a hydrated barrier saltcoated over the protein core. The advantage of such salt coating isimproved thermo-tolerance, improved storage stability and protectionagainst other feed additives otherwise having adverse effect on theenzyme and/or DFM.

Preferably, the salt used for the salt coating has a water activitygreater than 0.25 or constant humidity greater than 60% at 20° C.

Preferably, the salt coating comprises a Na₂SO₄.

The method of preparing a feed additive composition may also comprisethe further step of pelleting the powder. The powder may be mixed withother components known in the art. The powder, or mixture comprising thepowder, may be forced through a die and the resulting strands are cutinto suitable pellets of variable length.

Optionally, the pelleting step may include a steam treatment, orconditioning stage, prior to formation of the pellets. The mixturecomprising the powder may be placed in a conditioner, e.g. a mixer withsteam injection. The mixture is heated in the conditioner up to aspecified temperature, such as from 60-100° C., typical temperatureswould be 70° C., 80° C., 85° C., 90° C. or 95° C. The residence time canbe variable from seconds to minutes and even hours. Such as 5 seconds,10 seconds, 15 seconds, 30 seconds, 1 minutes, 2 minutes, 5 minutes, 10minutes, 15 minutes, 30 minutes and 1 hour.

It will be understood that the feed additive composition of the presentinvention is suitable for addition to any appropriate feed material.

As used herein, the term feed material refers to the basic feed materialto be consumed by an animal. It will be further understood that this maycomprise, for example, at least one or more unprocessed grains, and/orprocessed plant and/or animal material such as soybean meal or bonemeal.

As used herein, the term “feedstuff” refers to a feed material to whichone or more feed additive compositions have been added.

It will be understood by the skilled person that different animalsrequire different feedstuffs, and even the same animal may requiredifferent feedstuffs, depending upon the purpose for which the animal isreared.

Preferably, the feedstuff may comprise feed materials comprising maizeor corn, wheat, barley, triticale, rye, rice, tapioca, sorghum, and/orany of the by-products, as well as protein rich components like soybeanmean, rape seed meal, canola meal, cotton seed meal, sunflower seedmean, animal-by-product meals and mixtures thereof. More preferably, thefeedstuff may comprise animal fats and/or vegetable oils.

Optionally, the feedstuff may also contain additional minerals such as,for example, calcium and/or additional vitamins.

Preferably, the feedstuff is a corn soybean meal mix.

In one embodiment, preferably the feed is not pet food.

In another aspect there is provided a method for producing a feedstuff.Feedstuff is typically produced in feed mills in which raw materials arefirst ground to a suitable particle size and then mixed with appropriateadditives. The feedstuff may then be produced as a mash or pellets; thelater typically involves a method by which the temperature is raised toa target level and then the feed is passed through a die to producepellets of a particular size. The pellets are allowed to cool.Subsequently liquid additives such as fat and enzyme may be added.Production of feedstuff may also involve an additional step thatincludes extrusion or expansion prior to pelleting—in particular bysuitable techniques that may include at least the use of steam.

The feedstuff may be a feedstuff for a monogastric animal, such aspoultry (for example, broiler, layer, broiler breeders, turkey, duck,geese, water fowl), swine (all age categories), a pet (for example dogs,cats) or fish, preferably the feedstuff is for poultry.

In one embodiment the feedstuff is not for a layer.

By way of example only a feedstuff for chickens, e.g. broiler chickensmay be comprises of one or more of the ingredients listed in the tablebelow, for example in the %ages given in the table below:

Ingredients Starter (%) Finisher (%) Maize 46.2 46.7 Wheat Middlings 6.710.0 Maize DDGS 7.0 7.0 Soyabean Meal 48% CP 32.8 26.2 An/Veg Fat blend3.0 5.8 L-Lysine HCl 0.3 0.3 DL-methionine 0.3 0.3 L-threonine 0.1 0.1Salt 0.3 0.4 Limestone 1.1 1.1 Dicalcium Phosphate 1.2 1.2 PoultryVitamins and Micro-minerals 0.3 0.3

By way of example only the diet specification for chickens, such asbroiler chickens, may be as set out in the Table below:

Diet specification Crude Protein (%) 23.00 20.40 Metabolizable EnergyPoultry 2950 3100 (kcal/kg) Calcium (%) 0.85 0.85 Available Phosphorus(%) 0.38 0.38 Sodium (%) 0.18 0.19 Dig. Lysine (%) 1.21 1.07 Dig.Methionine (%) 0.62 0.57 Dig. Methionine + Cysteine (%) 0.86 0.78 Dig.Threonine (%) 0.76 0.68

By way of example only a feedstuff laying hens may be comprises of oneor more of the ingredients listed in the table below, for example in the%ages given in the table below:

Ingredient Laying phase (%) Maize 10.0 Wheat 53.6 Maize DDGS 5.0 SoybeanMeal 48% CP 14.9 Wheat Middlings 3.0 Soybean Oil 1.8 L-Lysine HCl 0.2DL-methionine 0.2 L-threonine 0.1 Salt 0.3 Dicalcium Phosphate 1.6Limestone 8.9 Poultry Vitamins and Micro-minerals 0.6

By way of example only the diet specification for laying hens may be asset out in the Table below:

Diet specification Crude Protein (%) 16.10 Metabolizable Energy Poultry2700 (kcal/kg) Lysine (%) 0.85 Methionine (%) 0.42 Methionine + Cysteine(%) 0.71 Threonine (%) 0.60 Calcium (%) 3.85 Available Phosphorus (%)0.42 Sodium (%) 0.16

By way of example only a feedstuff for turkeys may be comprises of oneor more of the ingredients listed in the table below, for example in the%ages given in the table below:

Phase 3 Phase 4 Ingredient Phase 1 (%) Phase 2 (%) (%) (%) Wheat 33.642.3 52.4 61.6 Maize DDGS 7.0 7.0 7.0 7.0 Soyabean Meal 48% CP 44.6 36.627.2 19.2 Rapeseed Meal 4.0 4.0 4.0 4.0 Soyabean Oil 4.4 4.2 3.9 3.6L-Lysine HCl 0.5 0.5 0.4 0.4 DL-methionine 0.4 0.4 0.3 0.2 L-threonine0.2 0.2 0.1 0.1 Salt 0.3 0.3 0.3 0.3 Limestone 1.0 1.1 1.1 1.0 DicalciumPhosphate 3.5 3.0 2.7 2.0 Poultry Vitamins 0.4 0.4 0.4 0.4 and Micro-minerals

By way of example only the diet specification for turkeys may be as setout in the Table below:

Diet specification Crude Protein (%) 29.35 26.37 22.93 20.00Metabolizable Energy Poultry 2.850 2.900 2.950 3.001 (kcal/kg) Calcium(%) 1.43 1.33 1.22 1.02 Available Phosphorus (%) 0.80 0.71 0.65 0.53Sodium (%) 0.16 0.17 0.17 0.17 Dig. Lysine (%) 1.77 1.53 1.27 1.04 Dig.Methionine (%) 0.79 0.71 0.62 0.48 Dig. Methionine + Cysteine (%) 1.121.02 0.90 0.74 Dig. Threonine (%) 1.03 0.89 0.73 0.59

By way of example only a feedstuff for piglets may be comprises of oneor more of the ingredients listed in the table below, for example in the%ages given in the table below:

Ingredient Phase 1 (%) Phase 2 (%) Maize 20.0 7.0 Wheat 25.9 46.6 Rye4.0 10.0 Wheat middlings 4.0 4.0 Maize DDGS 6.0 8.0 Soyabean Meal 48% CP25.7 19.9 Dried Whey 10.0 0.0 Soyabean Oil 1.0 0.7 L-Lysine HCl 0.4 0.5DL-methionine 0.2 0.2 L-threonine 0.1 0.2 L-tryptophan 0.03 0.04Limestone 0.6 0.7 Dicalcium Phosphate 1.6 1.6 Swine Vitamins and Micro-0.2 0.2 minerals Salt 0.2 0.4

By way of example only the diet specification for piglets may be as setout in the Table below:

Diet specification Crude Protein (%) 21.50 20.00 Swine Digestible Energy3380 3320 (kcal/kg) Swine Net Energy (kcal/kg) 2270 2230 Calcium (%)0.80 0.75 Digestible Phosphorus (%) 0.40 0.35 Sodium (%) 0.20 0.20 Dig.Lysine (%) 1.23 1.14 Dig. Methionine (%) 0.49 0.44 Dig. Methionine +Cysteine (%) 0.74 0.68 Dig. Threonine (%) 0.80 0.74

By way of example only a feedstuff for grower/finisher pigs may becomprises of one or more of the ingredients listed in the table below,for example in the %ages given in the table below:

Ingredient Grower/Finisher (%) Maize 27.5 Soyabean Meal 48% CP 15.4Maize DDGS 20.0 Wheat bran 11.1 Rice bran 12.0 Canola seed meal 10.0Limestone 1.6 Dicalcium phosphate 0.01 Salt 0.4 Swine Vitamins andMicro-minerals 0.3 Lysine-HCl 0.2 Vegetable oil 0.5

By way of example only the diet specification for grower/finisher pigsmay be as set out in the Table below:

Diet specification Crude Protein (%) 22.60 Swine Metabolizable Energy3030 (kcal/kg) Calcium (%) 0.75 Available Phosphorus (%) 0.29 DigestibleLysine (%) 1.01 Dig. Methionine + Cysteine (%) 0.73 Digestible Threonine(%) 0.66

Forms

The feed additive composition of the present invention and othercomponents and/or the feedstuff comprising same may be used in anysuitable form.

The feed additive composition of the present invention may be used inthe form of solid or liquid preparations or alternatives thereof.Examples of solid preparations include powders, pastes, boluses,capsules, pellets, tablets, dusts, and granules which may be wettable,spray-dried or freeze-dried. Examples of liquid preparations include,but are not limited to, aqueous, organic or aqueous-organic solutions,suspensions and emulsions.

In some applications, DFM or feed additive compositions of the presentinvention may be mixed with feed or administered in the drinking water.In one embodiment the dosage range for inclusion into water is about1×10³ CFU/animal/day to about 1×10¹⁰ CFU/animal/day, and more preferablyabout 1×10⁷ CFU/animal/day.

Suitable examples of forms include one or more of: powders, pastes,boluses, pellets, tablets, pills, capsules, ovules, solutions orsuspensions, which may contain flavouring or colouring agents, forimmediate-, delayed-, modified-, sustained-, pulsed- orcontrolled-release applications.

By way of example, if the composition of the present invention is usedin a solid, e.g. pelleted form, it may also contain one or more of:excipients such as microcrystalline cellulose, lactose, sodium citrate,calcium carbonate, dibasic calcium phosphate and glycine; disintegrantssuch as starch (preferably corn, potato or tapioca starch), sodiumstarch glycollate, croscarmellose sodium and certain complex silicates;granulation binders such as polyvinylpyrrolidone,hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),sucrose, gelatin and acacia; lubricating agents such as magnesiumstearate, stearic acid, glyceryl behenate and talc may be included.

Examples of nutritionally acceptable carriers for use in preparing theforms include, for example, water, salt solutions, alcohol, silicone,waxes, petroleum jelly, vegetable oils, polyethylene glycols, propyleneglycol, liposomes, sugars, gelatin, lactose, amylose, magnesiumstearate, talc, surfactants, silicic acid, viscous paraffin, perfumeoil, fatty acid monoglycerides and diglycerides, petroethral fatty acidesters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

Preferred excipients for the forms include lactose, starch, a cellulose,milk sugar or high molecular weight polyethylene glycols.

For aqueous suspensions and/or elixirs, the composition of the presentinvention may be combined with various sweetening or flavouring agents,colouring matter or dyes, with emulsifying and/or suspending agents andwith diluents such as water, propylene glycol and glycerin, andcombinations thereof.

Non-hydroscopic whey is often used as a carrier for DFMs (particularlybacterial DFMs) and is a good medium to initiate growth.

Bacterial DFM containing pastes may be formulated with vegetable oil andinert gelling ingredients.

Fungal products may be formulated with grain by-products as carriers.

In one embodiment preferably the feed additive composition according tothe present invention is not in the form of a microparticle system, suchas the microparticle system taught in WO2005/123034.

Dosing

The DFM and/or feed additive composition according to the presentinvention may be designed for one-time dosing or may be designed forfeeding on a daily basis.

The optimum amount of the composition (and each component therein) to beused in the combination of the present invention will depend on theproduct to be treated and/or the method of contacting the product withthe composition and/or the intended use for the same.

The amount of DFM and enzyme(s) used in the compositions should be asufficient amount to be effective and to remain sufficiently effectivein improving the performance of the animal fed feed products containingsaid composition.

This length of time for effectiveness should extend up to at least thetime of utilisation of the product (e.g. feed additive composition orfeed containing same).

The ratio of DFM to enzyme in the feed can be in the ranges given below:

DFM:phytase (CFU/FTU): In range from 2.5×10³ CFU DFM: 1 FTU enzyme to6.7×10⁶ CFU :1 FTU enzyme; preferably in the range from 3.8×10³ CFU DFM:1 FTU enzyme to 2.0×10⁵ CFU :1 FTU enzyme.

In one embodiment preferably the feedstuff comprises the following:

a phytase at (at least) 1500 FTU/kg of feed; and

Envivo Pro (DFM) at at least 7.5×10⁴ CFU/g to 3.0×10⁶ CFU/g of feed.

In one embodiment preferably the feedstuff comprises the following:

a phytase at (at least) 2000 FTU/kg of feed; and

Envivo Pro (DFM) at 7.5×10⁴ CFU/g to 3.0×10⁵ CFU/g of feed.

In one embodiment preferably the feedstuff comprises the following:

a phytase at (at least) 3000 FTU/kg of feed; and

Envivo Pro (DFM) at 7.5×10⁴ CFU/g to 3.0×10⁵ CFU/g of feed.

In one embodiment preferably the feedstuff comprises the following:

a phytase at (at least) 1500 FTU/kg of feed; and

DFM at at least 5×10⁴ CFU/g to 1×10⁷ CFU/g of feed.

In one embodiment preferably the feedstuff comprises the following:

a phytase at (at least) 2000 FTU/kg of feed; and

DFM at at least 5×10⁴ CFU/g to 1×10⁷ CFU/g of feed.

In one embodiment preferably the feedstuff comprises the following:

a phytase at (at least) 3000 FTU/kg of feed; and

DFM at at least 5×10⁴ CFU/g to 1×10⁷ CFU/g of feed.

In another embodiment the feedstuff comprises the following:

In a preferred embodiment the feed additive composition comprisessufficient enzyme and DFMs to dose the feedstuff as follows:

a phytase at (at least) 1500 FTU/kg (e.g. at (at least) 2000 FTU/kg) offeed; and Envivo Pro (DFM) at 75,0000CFU/g to 150,0000CFU/g of feed.

In a preferred embodiment the feed additive composition comprisessufficient enzyme and DFMs to dose the feedstuff as follows:

a phytase at (at least) 1500 FTU/kg (e.g. at (at least) 2000 FTU/kg) offeed; and

Envivo Pro (DFM) at 37,5000CFU/g to 75,0000CFU/g of feed.

Combination with Other Components

The DFM and enzyme(s) for use in the present invention may be used incombination with other components. Thus, the present invention alsorelates to combinations. The DFM in combination with a phytase may bereferred to herein as “the feed additive composition of the presentinvention”.

The combination of the present invention comprises the feed additivecomposition of the present invention (or one or more of the constituentsthereof) and another component which is suitable for animal consumptionand is capable of providing a medical or physiological benefit to theconsumer.

In one embodiment preferably the “another component” is not a furtherenzyme or a further DFM.

The components may be prebiotics. Prebiotics are typicallynon-digestible carbohydrate (oligo- or polysaccharides) or a sugaralcohol which is not degraded or absorbed in the upper digestive tract.Known prebiotics used in commercial products and useful in accordancewith the present invention include inulin (fructo-oligosaccharide, orFOS) and transgalacto-oligosaccharides (GOS or TOS). Suitable prebioticsinclude palatinoseoligosaccharide, soybean oligosaccharide, alginate,xanthan, pectin, locust bean gum (LBG), inulin, guar gum,galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS),non-degradable starch, lactosaccharose, lactulose, lactitol, maltitol,maltodextrin, polydextrose (i.e. Litesse®), lactitol, lactosucrose,soybean oligosaccharides, palatinose, isomalto-oligosaccharides,gluco-oligosaccharides and xylo-oligosaccharides, pectin fragments,dietary fibres, mannan-oligosaccharides.

Dietary fibres may include non-starch polysaccharides, such asarabinoxylans, cellulose and many other plant components, such asresistant dextrins, inulin, lignin, waxes, chitins, pectins,beta-glucans and oligosaccharides.

In one embodiment the present invention relates to the combination ofthe feed additive composition according to the present invention (or oneor more of the constituents thereof) with a prebiotic. In anotherembodiment the present invention relates to a feed additive compositioncomprising (or consisting essentially of or consisting of) a DFM incombination with a high dosage of phytase, and a prebiotic.

The prebiotic may be administered simultaneously with (e.g. in admixturetogether with or delivered simultaneously by the same or differentroutes) or sequentially to (e.g. by the same or different routes) thefeed additive composition (or constituents thereof) according to thepresent invention.

Other components of the combinations of the present invention includepolydextrose, such as Litesse®, and/or a maltodextrin and/or lactitol.These other components may be optionally added to the feed additivecomposition to assist the drying process and help the survival of DFM.

Further examples of other suitable components include one or more of:thickeners, gelling agents, emulsifiers, binders, crystal modifiers,sweeteners (including artificial sweeteners), rheology modifiers,stabilisers, anti-oxidants, dyes, enzymes, carriers, vehicles,excipients, diluents, lubricating agents, flavouring agents, colouringmatter, suspending agents, disintegrants, granulation binders etc. Theseother components may be natural. These other components may be preparedby use of chemical and/or enzymatic techniques.

In one preferred embodiment the DFM and/or enzyme for use in the presentinvention may be used in combination with one or more lipids.

For example, the DFM and/or enzyme for use in the present invention maybe used in combination with one or more lipid micelles. The lipidmicelle may be a simple lipid micelle or a complex lipid micelle.

The lipid micelle may be an aggregate of orientated molecules ofamphipathic substances, such as a lipid and/or an oil.

As used herein the term “thickener or gelling agent” refers to a productthat prevents separation by slowing or preventing the movement ofparticles, either droplets of immiscible liquids, air or insolublesolids. Thickening occurs when individual hydrated molecules cause anincrease in viscosity, slowing the separation. Gelation occurs when thehydrated molecules link to form a three-dimensional network that trapsthe particles, thereby immobilising them.

The term “stabiliser” as used here is defined as an ingredient orcombination of ingredients that keeps a product (e.g. a feed product)from changing over time.

The term “emulsifier” as used herein refers to an ingredient (e.g. afeed ingredient) that prevents the separation of emulsions. Emulsionsare two immiscible substances, one present in droplet form, containedwithin the other. Emulsions can consist of oil-in-water, where thedroplet or dispersed phase is oil and the continuous phase is water; orwater-in-oil, where the water becomes the dispersed phase and thecontinuous phase is oil. Foams, which are gas-in-liquid, andsuspensions, which are solid-in-liquid, can also be stabilised throughthe use of emulsifiers.

As used herein the term “binder” refers to an ingredient (e.g. a feedingredient) that binds the product together through a physical orchemical reaction. During “gelation” for instance, water is absorbed,providing a binding effect. However, binders can absorb other liquids,such as oils, holding them within the product. In the context of thepresent invention binders would typically be used in solid orlow-moisture products for instance baking products: pastries, doughnuts,bread and others.

“Carriers” or “vehicles” mean materials suitable for administration ofthe DFM and/or enzymes and include any such material known in the artsuch as, for example, any liquid, gel, solvent, liquid diluent,solubilizer, or the like, which is non-toxic and which does not interactwith any components of the composition in a deleterious manner.

The present invention provides a method for preparing a feed additivecomposition comprising admixing a DFM, and a phytase with at least onephysiologically acceptable carrier selected from at least one ofmaltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or awheat component, sucrose, starch, Na₂SO₄, Talc, PVA, sorbitol, benzoate,sorbiate, glycerol, sucrose, propylene glycol, 1,3-propane diol,glucose, parabens, sodium chloride, citrate, acetate, phosphate,calcium, metabisulfite, formate and mixtures thereof.

Examples of excipients include one or more of: microcrystallinecellulose and other celluloses, lactose, sodium citrate, calciumcarbonate, dibasic calcium phosphate, glycine, starch, milk sugar andhigh molecular weight polyethylene glycols.

Examples of disintegrants include one or more of: starch (preferablycorn, potato or tapioca starch), sodium starch glycollate,croscarmellose sodium and certain complex silicates.

Examples of granulation binders include one or more of:polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),hydroxypropylcellulose (HPC), sucrose, maltose, gelatin and acacia.

Examples of lubricating agents include one or more of: magnesiumstearate, stearic acid, glyceryl behenate and talc.

Examples of diluents include one or more of: water, ethanol, propyleneglycol and glycerin, and combinations thereof.

The other components may be used simultaneously (e.g. when they are inadmixture together or even when they are delivered by different routes)or sequentially (e.g. they may be delivered by different routes).

Preferably, when the feed additive composition of the present inventionis admixed with another component(s), the DFM remains viable.

In one embodiment preferably the feed additive composition according tothe present invention does not comprise chromium or organic chromium

Concentrates

The DFMs for use in the present invention may be in the form ofconcentrates. Typically these concentrates comprise a substantially highconcentration of a DFM.

Feed additive compositions according to the present invention may have acontent of viable cells (colony forming units, CFUs) which is in therange of at least 10⁴ CFU/g (suitably including at least 10⁵ CFU/g, suchas at least 10⁶ CFU/g, e.g. at least 10⁷ CFU/g, at least 10⁸ CFU/g) toabout 10¹⁰ CFU/g (or even about 10¹¹ CFU/g or about 10¹² CFU/g).

When the DFM is in the form of a concentrate the feed additivecompositions according to the present invention may have a content ofviable cells in the range of at least 10⁹ CFU/g to about 10¹² CFU/g,preferably at least 10¹⁰ CFU/g to about 10¹² CFU/g.

Powders, granules and liquid compositions in the form of concentratesmay be diluted with water or resuspended in water or other suitablediluents, for example, an appropriate growth medium such as milk ormineral or vegetable oils, to give compositions ready for use.

The DFM or feed additive composition of the present invention or thecombinations of the present invention in the form of concentrates may beprepared according to methods known in the art.

In one aspect of the present invention the enzymes or feed is contactedby a composition in a concentrated form.

The compositions of the present invention may be spray-dried orfreeze-dried by methods known in the art.

Typical processes for making particles using a spray drying processinvolve a solid material which is dissolved in an appropriate solvent(e.g. a culture of a DFM in a fermentation medium). Alternatively, thematerial can be suspended or emulsified in a non-solvent to form asuspension or emulsion. Other ingredients (as discussed above) orcomponents such as anti-microbial agents, stabilising agents, dyes andagents assisting with the drying process may optionally be added at thisstage.

The solution then is atomised to form a fine mist of droplets. Thedroplets immediately enter a drying chamber where they contact a dryinggas. The solvent is evaporated from the droplets into the drying gas tosolidify the droplets, thereby forming particles. The particles are thenseparated from the drying gas and collected.

Subject

The term “subject”, as used herein, means an animal that is to be or hasbeen administered with a feed additive composition according to thepresent invention or a feedstuff comprising said feed additivecomposition according to the present invention.

The term “subject”, as used herein, means an animal.

In one embodiment, the subject is a mammal, bird, fish or crustaceanincluding for example livestock or a domesticated animal (e.g. a pet).

In one embodiment the “subject” is livestock.

The term “livestock”, as used herein refers to any farmed animal.Preferably, livestock is one or more of ruminants such as cattle (e.g.cows or bulls (including calves)), mono-gastric animals such as poultry(including broilers, chickens and turkeys), pigs (including piglets),birds, aquatic animals such as fish, agastric fish, gastric fish,freshwater fish such as salmon, cod, trout and carp, e.g. koi carp,marine fish such as sea bass, and crustaceans such as shrimps, musselsand scallops), horses (including race horses), sheep (including lambs).In another embodiment the “subject” is a domesticated animal or pet oran animal maintained in a zoological environment.

The term “domesticated animal or pet or animal maintained in azoological environment” as used herein refers to any relevant animalincluding canines (e.g. dogs), felines (e.g. cats), rodents (e.g. guineapigs, rats, mice), birds, fish (including freshwater fish and marinefish), and horses.

In one embodiment the subject may be challenged by an enteric pathogen.

By way of example a subject may have one or more enteric pathogenspresent in its gut or digestive tract. For example a subject may haveone or more enteric pathogens in its gut or digestive tract at a levelwhich:

-   -   i) results in loss of performance of the animal and/or    -   ii) is at clinically relevant levels; or    -   iii) is at sub-clinical levels.

The enteric pathogen may be Clostridium perfringens for example.

Performance

As used herein, “animal performance” may be determined by the feedefficiency and/or weight gain of the animal and/or by the feedconversion ratio and/or by the digestibility of a nutrient in a feed(e.g. amino acid digestibility) and/or digestible energy ormetabolizable energy in a feed and/or by nitrogen retention and/or byanimals ability to avoid the negative effects of necrotic enteritisand/or by the immune response of the subject.

Preferably “animal performance” is determined by feed efficiency and/orweight gain of the animal and/or by the feed conversion ratio.

By “improved animal performance” it is meant that there is increasedfeed efficiency, and/or increased weight gain and/or reduced feedconversion ratio and/or improved digestibility of nutrients or energy ina feed and/or by improved nitrogen retention and/or by improved abilityto avoid the negative effects of necrotic enteritis and/or by animproved immune response in the subject resulting from the use of feedadditive composition of the present invention in feed in comparison tofeed which does not comprise said feed additive composition.

Preferably, by “improved animal performance” it is meant that there isincreased feed efficiency and/or increased weight gain and/or reducedfeed conversion ratio.

As used herein, the term “feed efficiency” refers to the amount ofweight gain in an animal that occurs when the animal is fed ad-libitumor a specified amount of food during a period of time.

By “increased feed efficiency” it is meant that the use of a feedadditive composition according the present invention in feed results inan increased weight gain per unit of feed intake compared with an animalfed without said feed additive composition being present.

Feed Conversion Ratio (FCR)

As used herein, the term “feed conversion ratio” refers to the amount offeed fed to an animal to increase the weight of the animal by aspecified amount.

An improved feed conversion ratio means a lower feed conversion ratio.

By “lower feed conversion ratio” or “improved feed conversion ratio” itis meant that the use of a feed additive composition in feed results ina lower amount of feed being required to be fed to an animal to increasethe weight of the animal by a specified amount compared to the amount offeed required to increase the weight of the animal by the same amountwhen the feed does not comprise said feed additive composition.

Nutrient Digestibility

Nutrient digestibility as used herein means the fraction of a nutrientthat disappears from the gastro-intestinal tract or a specified segmentof the gastro-intestinal tract, e.g. the small intestine. Nutrientdigestibility may be measured as the difference between what isadministered to the subject and what comes out in the faeces of thesubject, or between what is administered to the subject and what remainsin the digesta on a specified segment of the gastro intestinal tract,e.g. the ileum.

Nutrient digestibility as used herein may be measured by the differencebetween the intake of a nutrient and the excreted nutrient by means ofthe total collection of excreta during a period of time; or with the useof an inert marker that is not absorbed by the animal, and allows theresearcher calculating the amount of nutrient that disappeared in theentire gastro-intestinal tract or a segment of the gastro-intestinaltract. Such an inert marker may be titanium dioxide, chromic oxide oracid insoluble ash. Digestibility may be expressed as a percentage ofthe nutrient in the feed, or as mass units of digestible nutrient permass units of nutrient in the feed.

Nutrient digestibility as used herein encompasses starch digestibility,fat digestibility, protein digestibility, and amino acid digestibility.

Energy digestibility as used herein means the gross energy of the feedconsumed minus the gross energy of the faeces or the gross energy of thefeed consumed minus the gross energy of the remaining digesta on aspecified segment of the gastro-intestinal tract of the animal, e.g. theileum. Metabolizable energy as used herein refers to apparentmetabolizable energy and means the gross energy of the feed consumedminus the gross energy contained in the faeces, urine, and gaseousproducts of digestion. Energy digestibility and metabolizable energy maybe measured as the difference between the intake of gross energy and thegross energy excreted in the faeces or the digesta present in specifiedsegment of the gastro-intestinal tract using the same methods to measurethe digestibility of nutrients, with appropriate corrections fornitrogen excretion to calculate metabolizable energy of feed.

Nitrogen Retention

Nitrogen retention as used herein means as subject's ability to retainnitrogen from the diet as body mass. A negative nitrogen balance occurswhen the excretion of nitrogen exceeds the daily intake and is oftenseen when the muscle is being lost. A positive nitrogen balance is oftenassociated with muscle growth, particularly in growing animals.

Nitrogen retention may be measured as the difference between the intakeof nitrogen and the excreted nitrogen by means of the total collectionof excreta and urine during a period of time. It is understood thatexcreted nitrogen includes undigested protein from the feed, endogenousproteinaceous secretions, microbial protein, and urinary nitrogen.

Survival

The term survival as used herein means the number of subject remainingalive. The term “improved survival” may be another way of saying“reduced mortality”.

Carcass Yield and Meat Yield

The term carcass yield as used herein means the amount of carcass as aproportion of the live body weight, after a commercial or experimentalprocess of slaughter. The term carcass means the body of an animal thathas been slaughtered for food, with the head, entrails, part of thelimbs, and feathers or skin removed. The term meat yield as used hereinmeans the amount of edible meat as a proportion of the live body weight,or the amount of a specified meat cut as a proportion of the live bodyweight.

Weight Gain

The present invention further provides a method of increasing weightgain in a subject, e.g. poultry or swine, comprising feeding saidsubject a feedstuff comprising a feed additive composition according tothe present invention.

An “increased weight gain” refers to an animal having increased bodyweight on being fed feed comprising a feed additive composition comparedwith an animal being fed a feed without said feed additive compositionbeing present.

The increase in weight gain may be at least 0.5%, at least 1%, at least2%, at least 3%, at least 5%, at least 10%, at least 12%, at least 15%,at least 20% or at least 30%. In some embodiments the improvement may beat least 50% or at least 100%.

The increase in weight gain may be in respect to a control in which thefeed used does not comprise a DFM and high dosage (i.e. >1500 FTU/kgfeed) phytase. In another aspect the increase in weight gain may be withrespect to the use of a feed comprising another phytase wherein thephytase is used in a low dose e.g. <1000 FTU/kg feed.

Clinical or Subclinical Intestinal Disease Challenge

In one embodiment a clinical or subclinical intestinal disease challengeis present in the subject.

In one embodiment the clinical or subclinical intestinal diseasechallenge may be caused by any pathogen or any pathogenic bacteria, suchas Clostridium perfringens or E.coli.

The term “clinical disease” means a stage in the history of a pathologiccondition that begins with anatomic or physiologic changes that aresufficient to produce recognizable signs and symptoms of a disease.

The term “subclinical disease” means or relates to a stage in the courseof a disease before the symptoms are fist noted.

The intestinal disease may be any intestinal infection which the subjectcan suffer from.

In one example the intestinal disease may be caused by pathogenicorganisms, such as pathogenic bacteria.

In one embodiment the intestinal disease may be caused by one or more ofthe following organisms: pathogenic Clostridium spp, such as Clostridiumperfringens and/or Clostridium difficile, and/or E. coli and/orSalmonella spp and/or Campylobacter spp.

In one embodiment the intestinal disease may be caused by one or more ofthe following pathogens: Clostridium perfringens and/or Clostridiumdifficile and/or E. coli, preferably Clostridium perfringens and/orClostridium difficile, more preferably Clostridium perfringens.

Necrotic enteritis

Necrotic enteritis is an acute or chronic enterotoxemia seen inchickens, turkeys and ducks worldwide, caused by Clostridiumperfringens. Necrotic enteritis is often characterised by afibrino-necrotic enteritis, usually of the mid-small intestine.Mortality may be 5-50%, usually around 10%. Infection occurs byfaecal-oral transmission. Spores of the causative organism are highlyresistant. Predisposing factors include coccidiosis/coccidiasis, diet(high protein), in ducks possibly heavy strains, high viscosity diets(often associated with high rye and wheat inclusions in the diet),contaminated feed and/or water, other debilitating diseases. The presentinvention relates to increasing the subject's resistance to necroticenteritis. In other words, the present invention relates to avoiding orreducing the negative effect of necrotic enteritis.

The term “resistance to” as used herein may encompasses the term“tolerance of”. Therefore in one embodiment the subject may not beresistant to necrotic enteritis but the subject may be able to toleratethe necrotic enteritis, i.e. without negative effects on performance ofthe subject.

In one embodiment the present invention relates to a feed additivecomposition according to the present invention for treating orpreventing necrotic enteritis in a subject. Typically the subject willbe one which has been or will be challenged with Clostridium perfringensand/or Eimeria species. Such challenge may come from the environment orthe application of live microorganisms in the feed or drinking water,e.g. when live coccidia vaccines are used. In another embodiment thepresent invention relates to a feed additive composition for preventingand/or treating coccidiosis in a subject.

The present invention yet further provides a method of preventing and/ortreating necrotic enteritis and/or coccidiosis wherein an effectiveamount of a feed additive composition according to the present inventionis administered to a subject.

Immune Response

Immune response as used herein means one of the multiple ways in whichDFMs modulate the immune system of animals, including increased antibodyproduction, up-regulation of cell mediated immunity, up-regulation ofpro-inflammatory cytokines, and augmented toll-like receptor signalling.It is understood that immuno-stimulation of the gastro intestinal tractby DFMs may be advantageous to protect the host against disease, andthat immuno-suppression of the gastro intestinal tract may beadvantageous to the host because less nutrients and energy are used tosupport the immune function.

Preferably the immune response is a cellular immune response.

Preferably immune response is measure by looking at immune markers.

Pathogenic Bacteria

The term pathogenic bacteria as used herein means for example toxigenicclostridia species, e.g. Clostridium perfringens and/or E. coli and/orSalmonella spp and/or Campylobacter spp. In one embodiment thepathogenic bacteria may be Avian pathogenic E. coli species.

The present invention may reduce populations of pathogenic bacteria inthe gastrointestinal tract of a subject.

Nutrient Excretion

In one embodiment the present invention relates to reducing nutrientexcretion in manure. This has positive effects on reducing environmentalhazards. For example, in a preferred embodiment the present inventionrelates to reducing nitrogen and/or phosphorus content in the subject'smanure. This, therefore, reduces the amount of nitrogen and/orphosphorus in the environment, which can be beneficial.

Probiotic

For some applications, it is believed that the DFM in the composition ofthe present invention can exert a probiotic culture effect. It is alsowithin the scope of the present invention to add to the composition ofthe present invention further probiotic and/or prebiotics.

Here, a prebiotic is:

“a non-digestible food ingredient that beneficially affects the host byselectively stimulating the growth and/or the activity of one or alimited number of beneficial bacteria”.

The term “probiotic culture” as used herein defines live microorganisms(including bacteria or yeasts for example) which, when for exampleingested or locally applied in sufficient numbers, beneficially affectsthe host organism, i.e. by conferring one or more demonstrable healthbenefits on the host organism. Probiotics may improve the microbialbalance in one or more mucosal surfaces. For example, the mucosalsurface may be the intestine, the urinary tract, the respiratory tractor the skin. The term “probiotic” as used herein also encompasses livemicroorganisms that can stimulate the beneficial branches of the immunesystem and at the same time decrease the inflammatory reactions in amucosal surface, for example the gut.

Whilst there are no lower or upper limits for probiotic intake, it hasbeen suggested that at least 10⁶-10¹², preferably at least 10⁸-10⁹,preferably 10⁸-10⁹, cfu as a daily dose will be effective to achieve thebeneficial health effects in a subject.

Isolated

In one aspect, suitably the enzyme or DFM used in the present inventionmay be in an isolated form. The term “isolated” means that the enzyme orDFM is at least substantially free from at least one other componentwith which the enzyme or DFM is naturally associated in nature and asfound in nature. The enzyme or DFM of the present invention may beprovided in a form that is substantially free of one or morecontaminants with which the substance might otherwise be associated.Thus, for example it may be substantially free of one or morepotentially contaminating polypeptides and/or nucleic acid molecules.

Purified

In one aspect, preferably the enzyme and/or DFM according to the presentinvention is in a purified form. The term “purified” means that theenzyme and/or DFM is present at a high level. The enzyme and/or DFM isdesirably the predominant component present in a composition.Preferably, it is present at a level of at least about 90%, or at leastabout 95% or at least about 98%, said level being determined on a dryweight/dry weight basis with respect to the total composition underconsideration.

It is envisaged within the scope of the present invention that theembodiments of the invention can be combined such that combinations ofany of the features described herein are included within the scope ofthe present invention. In particular, it is envisaged within the scopeof the present invention that any of the therapeutic effects of thebacteria may be exhibited concomitantly.

The invention will now be described, by way of example only, withreference to the following Figures and Examples.

EXAMPLES Example 1

Methods

A total of 1600 one-day-old Cobb male chicks are purchased from acommercial hatchery. At study initiation, fifty males are allocated toeach treatment pen by blocks. The study consists of the followingtreatments (Table 1):

TABLE 1 Experimental design of Example 1. Clostridium Dietaryperfringens Treatment Challenge Phyzyme XP¹ DFM (CFU/g feed)² 1 No 500FTU/kg None 2 Yes 500 FTU/kg None 3 Yes 500 FTU/kg Enviva Pro (7.5 × 10⁴CFU/g) 4 Yes 3000 FTU/kg  Enviva Pro (7.5 × 10⁴ CFU/g) ¹Phyzyme XP is a6-phytase from E. coli provided by Danisco A/S. ²Enviva Pro ® iscombination of Bacillus subtilis strains Bs2084, LSSAO1 and 15AP4,provided by Danisco A/S.

Bird weights by pen are recorded at study initiation, 23 d, 35 d, andtermination (42d). The pen is the unit of measure. Broiler diets are fedas crumbles (starter) or pellets (grower and finisher). Diets met orexceeded NRC standards (Table 2). The mixer is flushed to prevent crosscontamination of diets. All treatment feeds are mixed using a Davis S-20mixer and pelleted using a California Pellet Mill (cold pellettemperature 65-70 C). Samples are collected from each treatment dietfrom the beginning, middle, and end of each batch and blended togetherto confirm enzyme activities and Enviva Pro presence in feed.

TABLE 2 Experimental diet composition of Example 1. Starter GrowerFinisher (0-23 d) (23-35 d) (35-42 d) Ingredient (%) Maize 53.62 57.8759.82 Maize DDGS 10.00 10.00 10.00 Soybean Meal 49% CP 26.93 23.97 21.36Ampro 55 5.00 5.00 5.00 Soy oil 2.07 0.91 1.74 Lysine 0.24 0.24 0.24DL-methionine 0.21 0.19 0.18 L-threonine 0.01 0.01 0.01 Salt 0.30 0.340.35 Limestone 1.04 1.07 0.94 Dicalcium phosphate 0.26 0.11 0.02 Vitaminand trace 0.33 0.33 0.33 mineral premix Calculated Nutrient Composition(%) CP 22.60 21.50 20.39 Energy, kcal/kg 3060 3025 3100 Digestiblelysine 1.36 1.26 1.21 Digestible methionine 0.58 0.61 0.53 Digestiblethreonine 0.83 0.83 0.80

Birds receive feed ad-libitum appropriate to the treatment from day 0 to42. Enzymes and Enviva Pro are provided by Danisco in the appropriatemixtures and levels for all experimental treatments. The pens arearranged within the facility to prevent direct contact in order to avoidcontamination. A change from starter to grower occurred on day 23.Grower diet is replaced with the finisher diet on day 35. At each feedchange, feeders are removed from pens by block, weighed back, emptied,and refilled with the appropriate treatment diet. On the final day ofthe study feed is weighed. Pens are checked daily for mortality. When abird is culled or found dead, the date and removal weight (kg) arerecorded. A gross necropsy is performed on all dead or culled birds todetermine the sex and probable cause of death. Signs of NecroticEnteritis are noted.

All pens had approximately 4 inches of built up litter with a coating offresh pine shavings. All birds are spray vaccinated prior to placementinto pens with a commercial coccidiosis vaccine (Coccivac-B). On days20, 21 and 22 all birds, except Treatment 1, are dosed with a brothculture of C. perfringens. A field isolate of C. perfringens known tocause NE and originating from a commercial broiler operation is utilizedas the challenge organism. Fresh inoculum is used each day. Thetitration levels are approximately 1.0×10⁸⁻⁹. Each pen receives the sameamount of inoculum. The inoculum is administered by mixing into the feedfound in the base of the tube feeder. On day 23, five birds from eachpen are selected, euthanized, group weighed, and examined for the degreeof presence of Necrotic Enteritis lesions. The scoring was based on a 0to 3 score, with 0 being normal and 3 being the most severe (0=none,1=mild, 2=moderate, 3=marked/severe; Hofacre et al., 2003 J. Appl.Poult. Res. 12:60-64). No concomitant drug therapy is used during thestudy.

Means are separated using pair wise t-tests. Significant differences areconsidered at P<0.05. Pens are used as the experimental unit.

Results

Body weight gain is significantly reduced by the C. perfringenschallenge as shown from d23 until the end of the trial (FIG. 1).Supplementation with Enviva Pro significantly improves body weight gainto the level of the positive control on each of the sample days. Howeverfurther supplementation with a high phytase dose (treatment 10) improvedbody weight to a level significantly greater than the positive controlat days 12, 23 and 35 and a numerically greater level at d42.

Feed conversion ratio (FCR) is significantly increased by the C.perfringens challenge as shown form d 23 until the end of the trial(FIG. 2). Addition of Enviva Pro to the diet lead to a significantreduction in FCR from the negative control. However, supplementationwith the high phytase dose reduces the FCR to a level the same as foundin the positive control.

There is a reduction in mortality observed with the supplementation ofEnviva Pro (FIG. 3), which is further numerically reduced with thesupplementation of the high phytase dose. This can be accounted for by areduction in the severity of the C. perfringens challenge, supported bythe numerically reduced lesion scores found when the high phytase doseis supplemented (FIG. 4), which was not significantly different from thelesion scores of the positive control.

There is a clear benefit of utilising a high phytase dose (3000 FTU/kg)in combination with Enviva Pro, demonstrated with reduced FCR andincreased body weight gain compared to using Enviva Pro with a standard(500 FTU) dose of phytase. These benefits are accounted for by reducedintestinal damage.

Example 2

Materials and Methods

A total of 1040 one-day-old Cobb×Cobb 500 male chicks are purchased froma commercial hatchery. At study initiation, ten males are randomlyallocated to Petersime battery cages according to the respectivetreatments by blocks. Only healthy birds are selected for theexperiment, and no birds are replaced throughout the course of thestudy. The study consists of the following treatments (Table 1):

TABLE 1 Experimental design of Example 2. Dietary Enviva Clostridium AvPPhytase Pro¹ Dietary perfringens level (Axtra 75,000 Treatment Challenge(%) Phy²) cfu/g feed 1. Non challenged No 0.4 0 No positive control 2.Challenged Yes 0.4 0 No positive control 3. Non challenged No 0.18 0 Nonegative control 4. Challenged negative Yes 0.18 0 No control (CNC) 5.CNC + 500 FTU Yes 0.18 500 No 6. CNC + 1500 FTU Yes 0.18 1500 No 7.CNC + 3000 FTU Yes 0.18 3000 No 8. CNC + 10000 FTU Yes 0.18 10,000 No 9.CNC + Enviva Yes 0.18 0 Yes Pro (EP) 10. CNC + 500 Yes 0.18 500 YesFTU + EP 11. CNC + 1500 Yes 0.18 1500 Yes FTU + EP 12. CNC + 3000 Yes0.18 3000 Yes FTU + EP 13. CNC + 10000 Yes 0.18 10,000 Yes FTU +EP¹Enviva Pro is combination of Bacillus subtilis strains Bs2084, LSSAO1and 15AP4, provided by Danisco A/S, dosed at 75,000 CFU/g of feed.²AxtraPhy ™ is a 6-phytase from Buttiauxella, provided by Danisco A/S

Bird weights are recorded at study initiation (d 0) and on days 13, 21and study termination (d 28). The cage is the experimental unit. Dietsare fed in mash form, and are formulated to meet or exceed NRC standards(Table 2). The mixer is flushed to prevent cross contamination betweenrations. Samples are collected from each treatment diet from thebeginning, middle and end of each batch and are mixed together foranalysis of enzyme activity and DFM presence in feed.

TABLE 2 Experimental diet composition of Example 2. Starter Grower (PC)Grower (NC) (0 to 9 (10 to 28 (10 to 28 days) days) days) Ingredient (%)Maize 46.59 54.94 56.69 Maize DDGS 7.00 7.00 7.00 Soybean Meal 48% CP32.67 25.64 25.33 Rice Bran 5.00 5.00 5.00 Pig/Poultry Fat 4.03 3.112.53 Lysine 0.37 0.41 0.42 DL-methionine 0.36 0.32 0.31 L-threonine 0.150.15 0.16 Salt 0.38 0.35 0.35 Limestone 0.88 0.96 1.38 Dicalciumphosphate 2.08 1.60 0.33 Vitamin and trace 0.50 0.50 0.50 mineral premixCalculated Nutrient Composition (%) CP 22.66 20.00 20.00 Energy, kcal/kg3035 3059 3059 Digestible lysine 1.27 1.14 1.14 Digestible methionine0.66 0.60 0.60 Digestible threonine 0.80 0.72 0.72

All birds are fed a “commercial style” ration until day 9; from day 10the treatment rations are fed. At the feed change, feeders are removedfrom pens, weighed back, emptied, and refilled with the appropriatetreatment diet. On the final day of the study, feed is weighed. Pens arechecked daily for mortality. When a bird is culled or found dead, thedate and removal weight (kg) are recorded. A gross necropsy is performedon all dead or culled birds to determine the probable cause of death.Signs of Necrotic Enteritis are recorded. Ad-libitum feed of theappropriate treatment is available for the birds throughout the durationof the study.

Disease induction is according to the research site standard operatingprocedure (SOP). Briefly; on day 13, all birds are orally inoculatedwith Eimeria maxima, with inocula containing approximately 5,000 oocystsper bird. On day 18, all birds apart from treatments 1 and 3, receive abroth culture of Clostridium perfringens containing approximately 10⁸cfu/ml. All treated pens receive the same amount of inocula. Fresh C.perfringens inoculum is administered once daily for three days (days 18,19 and 20) by mixing into the feed in the base of the feeders. On day21, three birds from each pen are selected, euthanized, group weighed,and examined for the degree of presence of Necrotic Enteritis lesions.The scoring is based on a 0 to 3 score, with 0 being normal and 3 beingthe most severe (0=none, 1=mild, 2=moderate, 3=marked/severe; Hofacre etal., 2003 J. Appl. Poult. Res. 12:60-64). No concomitant drug therapy isused during the study.

For performance data, the effects of treatment are tested using ANOVA,and means are separated using pair wise t-tests using JMP software.Significant differences are considered at P<0.05. Cages are used as theexperimental unit. For pH and organ weight data, each bird is anexperimental unit.

In order to test interactions, a subset of treatments (treatments 4 to13) are analysed with ANOVA using a factorial arrangement that includedthe main effects of phytase and Enviva Pro, as well as theirinteraction.

Results

The C. perfringens challenge significantly reduce bird performance interms of both body weight gain (BWG) and feed conversion ratio (FCR), atboth d 21 and d 28, compared to the unchallenged control (T1 vs. T2, T3vs. T4) (Table 3). The reduction in dietary P has no effect on BWG ateither d 21 or d 28 in the absence of the C. perfringens challenge.There is however, a noticeable numerical reduction in BWG between thehigh and low P diets in the presence of the C. perfringens challenge.These observations are consistent with the data for FCR.

When phytase is supplemented alone, there are variable effects on birdperformance; however, in all cases, there is a numerical improvement inday 21 BWG to a level higher than that of the challenged low P diet(T4). Results obtained indicate that high phytase doses do not furtherimprove bird performance in the conditions of a Necrotic Enteritischallenge; as the lowest BWG of the phytase only treatments is found atthe 10,000 FTU dose of phytase (T8), which is not significantlydifferent than the challenged low P diet (T4). Likewise, at day 28, whenphytase is supplemented alone there is a numerical reduction in BWG atphytase levels greater than 1500 FTU.

When Enviva Pro is supplemented alone (T9) there is a significantimprovement in BWG at day 21 from the challenged low P diet (T4) and afurther improvement to a level not significantly different to theunchallenged low P diet by day 28. In terms of FCR, there is asignificant reduction from the challenged control with Enviva Prosupplementation, but not enough to completely counteract the negativeeffects of the challenge.

In combination, high doses of Phytase and Enviva Pro result in a moreuniform response in terms of BWG, compared to when Phytase issupplemented alone, at both day 21 and day 28. In terms of FCR, there isa synergistic effect of the combination at day 21 (FIG. 5). When phytaseis supplemented alone, there is an increase in performance (reduction inFCR) at the 500, 1500 (T7) and 3000 FTU/kg levels and a reduction inperformance when phytase dose is exceeded in the conditions of aNecrotic Enteritis challenge. However, when the probiotic is added incombination, there consistently is a numerical reduction in the FCRcompared to the phytase only treatments up to and including the level of3000 FTU (T12). However at 10,000 FTU (T13), there is a largesignificant reduction which results in the lowest FCR of all of thetreatment groups. When specifically investigating the effects of thecombination (i.e. excluding positive controls T1, T2 and T3) there is asignificant effect of the combination on day 21 FCR (P=0.0319) (Table4).

The reduction in dietary AvP causes significant reductions in tibia ash(Table 3), which is an indicator of phosphorus bioavailability. The C.perfringens challenge results in small numerical reductions in tibia ashcontent, which is likely due to the intestinal damage caused by theinfection reducing the ability of the gut to absorb nutrientsefficiently. Supplementation with phytase alone results in restorationof the tibia ash % to a level not significantly different to theunchallenged high P diet (T1). When Enviva Pro is supplemented alone(T9), there is a numerical increase in tibia ash % to a level greaterthan the unchallenged low P diet (T3), which demonstrates the ability ofthe product to counter the effects of the C. perfringens challenge interms of reductions in tibia ash, which could suggest restoration of gutfunction. When the combination was supplemented, there is a doseresponse to phytase reaching a level greater than the unchallenged highP diet (T1) at 3000 FTU, such an improvement is not observed whenphytase was supplemented alone

TABLE 3 The effect of Treatment on performance parameters; feed intake,body weight gain, feed conversion ratio and bone ash data at d21 FeedIntake Body Weight Gain Feed Conversion Ratio % Bone Dietary Treatmentd21 d28 d21 d28 d21 d28 Ash  1. Non challenged 6.587 e 9.829 ab 0.454 a0.943 ab 1.453 g 1.484 h 16.03 Ab positive control  2. Challenged 7.390a 8.374 de 0.410 bcd 0.828 ef 1.836 ab 1.780 ab 15.53 Bc positivecontrol  3. Non challenged 6.903 bcde 10.489 a 0.463 a 0.969 a 1.501 g1.507 gh 13.95 Ef negative control  4. Challenged 7.319 ab 8.887 cd0.387 d 0.796 f 1.893 a 1.838 a 13.71 F negative control (CNC)  5. CNC +500 7.278 abc 8.433 cde 0.424 b 0.884 bcde 1.731 cde 1.662 cde 15.55 abcFTU  6. CNC + 1500 7.003 abcde 7.923 e 0.397 bcd 0.899 bcd 1.780 bcd1.742 bc 15.57 abc FTU  7. CNC + 3000 7.175 abcd 8.228 de 0.414 bcd0.869 cde 1.726 cde 1.716 bc 15.68 abc FTU  8. CNC + 10000 7.317 ab8.118 de 0.393 cd 0.848 def 1.864 ab 1.721 bc 16.18 ab FTU  9. CNC +Enviva 7.435 a 9.345 bc 0.422 b 0.908 abcd 1.801 bc 1.688 cd 14.19 efPro (EP) 10. CNC + 500 6.833 cde 8.094 de 0.418 bc 0.938 ab 1.651 ef1.600 def 14.48 de FTU + EP 11. CNC + 1500 6.874 bcde 8.057 de 0.421 bc0.931 abc 1.661 ef 1.493 gh 15.03 cd FTU + EP 12. CNC + 3000 7.003 abcde8.028 de 0.420 bc 0.936 ab 1.706 def 1.546 fgh 16.27 a FTU + EP 13.CNC + 10000 6.782 de 8.587 cde 0.421 bc 0.905 abcd 1.633 f 1.566 efg16.00 ab FTU + EP Standard Error 0.182    0.348    0.0107   0.0237  0.0334   0.0343   0.269  Effect tests Treatment 0.0086 <0.0001 <0.0001<0.0001 <0.0001 <0.0001 <0.0001

TABLE 4 Significant effect of the combination of Phytase + Enviva Pro onday 21 FCR. Dietary Treatment FCR (0-21 d) 4. Challenged positivecontrol (CNC) 1.893 a  5. CNC + 500 FTU 1.731 cde 6. CNC + 1500 FTU1.780 bcd 7. CNC + 3000 FTU 1.726 cde 8. CNC + 10000 FTU 1.864 ab  9.CNC + Enviva Pro (EP) 1.801 abc 10. CNC + 500 FTU + EP 1.651 ef   11.CNC + 1500 FTU + EP 1.661 ef   12. CNC + 3000 FTU + EP 1.706 def  13.CNC + 10000 FTU + EP 1.633 f  Standard Error 0.0328   Effect testsPhytase 0.0001   Enviva Pro <.0001   Phytase*Enviva Pro 0.0319  

The C. perfringens challenge has a significant effect on mortality andlesion scores (Table 4). The lack of mortality and absence of lesionscores in the unchallenged groups indicates there is no contaminationbetween challenged and unchallenged groups. There is no significantdifference between the Phytase and EnvivaPro+Phytase at any level ofphytase in terms of lesions scores (T4-8 vs. T9-13). However, there is anumerical increase in mortality above the level of the challenged low Pdiet in the Phytase only treatments for the 1500 (T6) and 3000 FTU (T7)treatments. The addition of EnvivaPro leads to a significant reductionin mortality from the challenged low P diet and also reduced thevariation in mortality rates between the phytase doses in combinationcompared to the Phytase only treatments.

TABLE 5 The effect of Treatment on Mortality (%) and Necrotic enteritis(NE) lesion scores. Dietary NE lesion % NE Treatment Score Mortality 1.Non challenged positive control 0.000 e  0 d 2. Challenged positivecontrol  0.830 abcd 28.8 a 3. Non challenged negative control 0.000 e  0d 4. Challenged negative control (CNC) 0.580 cd  27.5 ab 5. CNC + 500FTU  0.830 abcd 20 bc 6. CNC + 1500 FTU 1.340 a  31.3 a 7. CNC + 3000FTU  0.790 bcd 31.3 a 8. CNC + 10000 FTU 1.250 ab  27.5 ab 9. CNC +Enviva Pro (EP) 0.390 de 16.3 c 10. CNC + 500 FTU + EP  0.980 abc 18.8 c11. CNC + 1500 FTU + EP 1.160 ab 17.5 c 12. CNC + 3000 FTU + EP  0.890abcd 17.5 c 13. CNC + 10000 FTU + EP  1.080 abc 17.5 c Standard Error0.193    0.294 Effect tests Treatment <0.0001    <0.0001

Example 3

Methods

A total of 1080 one day old chicks are purchased from a commercialhatchery. At study initiation 15 chicks are randomly allocated to eachpen by blocks. The study consists of the following treatments (table 1)and there are 8 replicate pens per treatment.

TABLE 1 Experimental design Av. P Axtra Dietary (Starter/ DFM (CFU/gPhy² Treatment Finisher) feed)¹ (FTU/kg) 1. Positive control 0.45/0.42No 0 2. Negative control (NC) 0.20/0.20 No 0 3. NC + 500 FTU 0.20/0.20No 500 4. NC + 1500 FTU 0.20/0.20 No 1500 5. NC + 3000 FTU 0.20/0.20 No3000 6. NC + Enviva Pro (EP) 0.20/0.20 Enviva Pro 0 (7.5 × 10⁴ cfu/g) 7.NC + 500 FTU + EP 0.20/0.20 Enviva Pro 500 (7.5 × 10⁴ cfu/g) 8. NC +1500 FTU + EP 0.20/0.20 Enviva Pro 1500 (7.5 × 10⁴ cfu/g) 9. NC + 3000FTU + EP 0.20/0.20 Enviva Pro 3000 (7.5 × 10⁴ cfu/g) ¹Enviva Pro ® iscombination of Bacillus subtilis strains Bs2084, LSSAO1 and 15AP4,provided by Danisco A/S. ²Axtra Phy is a 6-phytase from Butiauxellaavailable from Danisco Animal Nutrition

Bird weights are recorded at study initiation (day 0), day 6, day 21 andstudy termination (day 35). The pen is the unit of measure. Diets areformulated to meet NRC guidelines (Table 2). All treatment feeds aremixed using a Davis S-20 mixer. The mixer is flushed between diets toprevent cross contamination of diets. Samples are collected from eachtreatment diet form the beginning, middle and end of each batch, pooled,and analysed to confirm enzyme activity and DFM presence in feed. Dietsare fed in mash form.

All pens have approximately 4 inches of built up litter with a coatingof fresh pine shavings. All birds receive a commercial coccidiosisvaccine orally prior to placement into pens.

TABLE 2 Experimental diet composition Grower Finisher Starter PC NC PCNC Ingredient (%) Maize 52.42 56.39 58.56 60.48 62.29 US Maize DDGS 7 77 7 7 Soybean Meal 48% CP 26.13 22.13 21.93 18.12 17.96 Canola Meal 3 33 3 3 Feather Meal 3 3 3 3 3 Pig/Poultry Fat 3.57 4.26 3.49 4.45 3.81L-Lysine HCl 0.52 0.43 0.43 0.38 0.39 DL-methionine 0.33 0.26 0.26 0.210.21 L-threonine 0.13 0.08 0.08 0.06 0.05 Salt 0.35 0.35 0.35 0.35 0.35Limestone 0.97 0.76 1.02 0.75 1.03 Dicalcium Phosphate 2.09 1.84 0.381.70 0.42 Poultry Vits/TE's 0.5 0.5 0.5 0.5 0.5 Calculated NutrientComposition Crude Protein (%) 23.0 21.3 21.4 19.6 19.7 Energy MJ/Kg12.65 13 13 13.2 13.2 Dig. Lysine (%) 1.27 1.1 1.1 0.97 0.97 Dig.Methionine + Cystine (%) 0.94 0.84 0.84 0.76 0.76 Dig. Threonine (%)0.83 0.73 0.73 0.65 0.65 Available P (%) 0.5 0.45 0.2 0.42 0.2

All birds are fed a commercial style starter ration from day 0-6. Fromday 7 grower diets appropriate to the treatment are fed until day 21.Finisher diets appropriate to treatment are fed from day 21 to studytermination (day 35). At the feed change, feeders are removed from pens,weighed back, emptied, and refilled with the appropriate treatment diet.On the final day of the study, feed is weighed. Pens are checked dailyfor mortality. When a bird is culled or found dead, the date and removalweight (kg) are recorded.

For performance data, means are separated using pair wise t-tests.Significant differences are considered at P<0.05. Pens are used as theexperimental unit.

Results

Increasing the dose of phytase alone causes no significant improvementin feed conversion ratio (FCR) at 35 days of age (FIG. 6). The additionof Enviva Pro at all levels of phytase reduces FCR. As phytase doseincreases so does the benefit from adding Enviva Pro. The highest levelof phytase inclusion creates the largest difference in FCR between thediets with and without Enviva Pro.

TABLE 3 Performance results Body weight Feed intake FCR Dietary gain(g/bird) (g/bird) (g BWG/g FI) Treatment Day 35 Day 6-35 Day 6-35 1.Positive control 1961.1^(a) 3225.5^(a) 1.856^(ab) 2. Negative control(NC) 1785.9^(b) 2953.3^(bc) 1.889^(a ) 3. NC + 500 FTU 1976.8^(a)3158.4^(a) 1.812^(bc) 4. NC + 1500 FTU 2009.4^(a) 3196.9^(a) 1.809^(bc)5. NC + 3000 FTU 2007.3^(a) 3175.6^(a) 1.808^(bc) 6. NC + Enviva Pro(EP) 1757.1^(b) 2787.9^(c)  1.829^(abc) 7. NC + 500 FTU + EP 1947.8^(a)3065.1^(ab) 1.786^(bc) 8. NC + 1500 FTU + EP 2020.5^(a) 3164.9^(a)1.770^(cd) 9. NC + 3000 FTU + EP 2026.1^(a) 3049.0^(ab) 1.711^(d)  SEM34.33 64.19 0.0252  Source of Variation <0.0001 0.0001 0.0006  Treatment

At higher doses of phytase, there is a numerical increase in body weightgain (BWG) when Enviva Pro is supplemented compared to when phytase issupplemented alone (Table 3). This effect is not seen at the lowest dose(500 FTU) of phytase.

Example 4

Materials and Methods

One day old Cobb male chicks are purchased from a commercial hatchery.At study initiation, ten males are randomly allocated to each batterycage by blocks. There are eight replicate cages per treatment. The studyconsists of the following treatments (Table 1).

TABLE 1 Experimental Design of Example 4 Clostridium Available P AmountDFM perfringens in diet (FTU/kg (CFU/g Treatment Challenge (%) Phytasefeed) feed)² 1 No 0.4 None 0 None 2 Yes 0.4 None 0 None 3 No 0.18 None 0None 4 Yes 0.18 None 0 None 5 Yes 0.18 HiPhos¹ 500 None 6 Yes 0.18HiPhos¹ 1500 None 7 Yes 0.18 HiPhos¹ 3000 None 8 Yes 0.18 None 0 EnvivaPro (7.5 × 10⁴ FTU/g) 9 Yes 0.18 HiPhos¹ 500 Enviva Pro (7.5 × 10⁴FTU/g) 10 Yes 0.18 HiPhos¹ 1500 Enviva Pro (7.5 × 10⁴ FTU/g) 11 Yes 0.18HiPhos¹ 3000 Enviva Pro (7.5 × 10⁴ FTU/g) ¹HiPhos is Ronozyme HiPhos andcontains a phytase from Citrobacter braakii and is available fromDSM/Novozymes ²Enviva Pro ® is combination of Bacillus subtilis strainsBs2084, LSSAO1 and 15AP4, provided by Danisco A/S.

Bird weights are recorded at study initiation (0 d), day 21 andtermination (28 d). The cage is the unit of measure. Diets meet orexceed NRC standards (Table 2). All treatment feeds are mixed using aDavis S-20 mixer. The mixer is flushed between diets to prevent crosscontamination of diets. Samples are collected from each treatment dietfrom the beginning, middle, and end of each batch and blended togetherto confirm enzyme activities and Enviva Pro presence in feed.

TABLE 2 Experimental diet composition of Example 4 Starter Grower (PC)Grower (NC) (0 to 9 (10 to 28 (10 to 28 days) days) days) Ingredient (%)Maize 46.59 54.94 56.69 Maize DDGS 7.00 7.00 7.00 Soybean Meal 48% CP32.67 25.64 25.33 Rice Bran 5.00 5.00 5.00 Pig/Poultry Fat 4.03 3.112.53 Lysine 0.37 0.41 0.42 DL-methionine 0.36 0.32 0.31 L-threonine 0.150.15 0.16 Salt 0.38 0.35 0.35 Limestone 0.88 0.96 1.38 Dicalciumphosphate 2.08 1.60 0.33 Vitamin and trace 0.50 0.50 0.50 mineral premixCalculated Nutrient Composition (%) CP 22.66 20.00 20.00 Energy, kcalME/kg 3035 3059 3059 Digestible lysine (%) 1.27 1.14 1.14 Digestiblemethionine (%) 0.66 0.60 0.60 Digestible threonine (%) 0.80 0.72 0.72

All birds are fed a commercial pre-starter ration until day 9; from day10 the treatment rations are fed. At the feed change, feeders areremoved from cages, weighed back, emptied, and refilled with theappropriate treatment diet. On the final day of the study, feed isweighed. Cages are checked daily for mortality. When a bird is culled orfound dead, the date and removal weight (kg) are recorded. A grossnecropsy is performed on all dead or culled birds to determine theprobable cause of death. Signs of Necrotic Enteritis are recorded.Ad-libitum feed of the appropriate treatment is available for the birdsthroughout the duration of the study.

Disease induction is according to the research site standard operatingprocedure (SOP). Briefly; on day 13, all birds we orally inoculate withEimeria maxima, with inocula containing approximately 5,000 oocysts perbird. On day 18, all birds apart from treatments 1 and 3, receive abroth culture of Clostridium perfringens containing approximately108cfu/ml. All treated pens receive the same amount of inocula. Fresh C.perfringens inoculum is administered once daily for three days (days 18,19 and 20) by mixing into the feed in the base of the feeders. On day21, three birds from each pen are selected, euthanized, group weighed,and examined for the degree of presence of Necrotic Enteritis lesions.The scoring is based on a 0 to 3 score, with 0 being normal and 3 beingthe most severe (0=none, 1=mild, 2=moderate, 3=marked/severe; Hofacre etal., 2003 J. Appl. Poult. Res. 12:60-64). No concomitant drug therapy isused during the study.

The pH of ileal digesta of 2 birds per cage is measured on day 21. ThepH of the digesta is determined by inserting a Sensorex spear tippiercing pH probe into the respective sections.

In both birds, the gizzard are also removed and weighed. On day 21, theright tibias of the two birds are removed, dried overnight at 100° C.and weighed; samples are then fat-extracted and burned in a mufflefurnace at 600° C. for 16 hours to determine the fat free bone ash. Thepercentage bone ash is then calculated as the ratio of remaining ashweight to the dry-fat free bone weight multiplied by 100.

Statistical Analysis

For performance data, means are separated using pair wise t-tests.Significant differences are considered at P<0.05. Cages are used as theexperimental unit.

Results:

Increasing the dose of Hi Phos causes an increase in feed conversionratio at 21days of age (FIG. 7; more feed per unit of body weight gain)in a Necrotic Enteritis challenge situation. The addition of Enviva Proat all levels of inclusion of phytase cause a decrease, improving FCR.At the highest level of phytase inclusion (3000 FTU/kg) the increase inFCR seen over the 1500 FTU/kg dose is increasingly attenuated by theaddition of Enviva Pro.

TABLE 3 Performance results from Example 4 Body Weight Feed Intake FCRGain Dietary 0-21 d 0-21 d 0-21 d Treatment (g/bird) (g/g) (g/bird) PC524.052bc 1.390d 378ab  Unchallenged NC 597.000ab 1.450d 412a Challenged (Cc) PC 480.980c 1.619c 298d  Challenged (Cc) NC 558.952ab1.716a 326cd  Cc 500 HiPhos 600.161a  1.657abc 366abc Cc 1500 HiPhos611.731a  1.700ab 360bc  Cc 3000 HiPhos 602.466a 1.727a 350bc  Cc +Enviva Pro (EP) 585.231ab  1.626bc 361abc Cc 500 HiPhos + EP 554.078abc1.599c 347bcd Cc 1500 HiPhos + EP 595.992ab  1.638bc 365abc Cc 3000HiPhos + EP 611.336a  1.636bc 374abc SEM 26.456 0.027  18  Source ofVariation <0.01 <0.01    <0.01 Treatment

The body weight gain effect of Enviva Pro on top of phytase is moreevident at high phytase doses (3000 FTU/kg), where the body weight gainwas not significantly different compared to that in the unchallengedcontrol treatment.

The effect of phytase on tibia ash percentage is not significantlydifferent to the negative challenged control, suggesting that phytasedoes not have a significant effect on tibia ash under challengesituations, even at high doses (FIG. 8). The addition of Enviva Prohowever acts to significantly improve tibia ash percentage to bring itclose to that seen in the positive unchallenged control at the highestdose of phytase (3000 FTU/kg) as seen in Table 4.

TABLE 4 Other physiological measurements from Example 5 Gizzard LesionTibia Ileal weight Score Dietary Ash pH at 21 d Mortality (1-3 Treatment(%) 21 d (g/bird) (%) score) PC 16.520a 6.116bcd 24.356a  0.000e 0.000cUnchallenged NC  14.899cde 6.326abc 24.650a  0.000e 0.000c Challenged(Cc) PC  15.742abc 6.298abc 24.058ab 15.000a   0.417ab Challenged (Cc)NC 14.423e 6.074cd  20.275b  11.250ab  0.333abc Cc 500 HiPhos 14.526e6.420abc 22.392ab  5.000cde  0.250abc Cc 1500 HiPhos  14.724de 6.313abc23.123ab  7.500bcd 0.542a Cc 3000 HiPhos 14.597e 6.540a  20.261b  8.750bc 0.542a Cc + Enviva Pro (EP)  14.957cde 5.889d   23.506ab 3.750cde  0.375ab Cc 500 HiPhos + EP 16.560a 6.398abc 21.211ab  2.500de0.500a Cc 1500 HiPhos + EP  15.567bcd 6.486ab  21.480ab  6.250bcd 0.125bc Cc 3000 HiPhos + EP  16.172ab 6.389abc 20.770ab  7.500bcd 0.375ab SEM 0.329 0.132   1.431  1.890  0.119  Source of Variation<0.001  <0.05      0.226596 <0.001  <0.01   Treatment

High levels of phytase (3000 FTU/kg) increase ileal pH at 21 d over therelevant control, and the addition of Enviva Pro act to reduce it, withthe lowest pH seen with the Enviva Pro alone treatment. The lowestgizzard weights are seen in the challenged negative control and thehighest dose of Hi Phos (3000 FTU/kg). The addition of Enviva Pronumerically increase gizzard weight at the 3000 FTU/kg dose but notsignificantly.

The challenge increases mortality significantly, with the addition ofphytase acting to reduce it. The combination with Enviva Pro acted toreduce it further.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and biotechnology or related fields areintended to be within the scope of the following claims.

1. A method for improving the performance of a subject or for improvingdigestibility of a raw material in a feed (e.g. nutrient digestibility,such as amino acid digestibility), or for improving nitrogen retention,or for improving dietary phosphorus absorption and retention, or forimproving the efficacy of the phytase, or for improving the subject'sresistance to necrotic enteritis or for improving feed conversion ratio(FCR) or for improving weight gain in a subject or for improving feedefficiency in a subject or for modulating (e.g. improving) the immuneresponse of the subject or for reducing populations of pathogenicbacteria in the gastrointestinal tract of a subject, or for reducingnutrient excretion in manure, which method comprising administering to asubject at least one direct fed microbial in combination with a phytase,wherein the phytase is administered to the subject at a dosage of morethan about 1500 FTU/kg feed.
 2. (canceled)
 3. A method according toclaim 1 wherein the direct fed microbial is an antipathogen direct fedmicrobial.
 4. A method according to claim 1 wherein the direct fedmicrobial is a viable bacterium.
 5. A method according to claim 1wherein the phytase is administered to the subject at a dosage of morethan about 2000 FTU/kg feed.
 6. A method according to claim 1 whereinthe phytase is administered to the subject at a dosage of more thanabout 3000 FTU/kg feed.
 7. A method according to claim 1 wherein thecomposition comprises as least three direct fed microbials.
 8. A methodaccording to claim 1 wherein the direct fed microbial is a strain whichinhibits intestinal establishment of pathogenic microorganisms (such asClostridium perfringens and/or E.coli)
 9. A method according to claim 1wherein the direct fed microbial comprises a bacterium from one or moreof the following genera: Bacillus, Enterococcus, Pediococcus,Saccharomyces, Bifidobacterium, Propionibacterium, Lactobacillus,Lactococcus, Aspergillus and combinations thereof.
 10. A methodaccording to claim 1 wherein the direct fed microbial comprises abacterium from one or more of the following species: Bacillus subtilis,Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus,Enterococcus faecium, Pediococcus acidilactici, Saccharomycescerevisiae, Bifidobacterium animalis spp animalis, Lactobacillusreuteri, Lactobacillus rhamnosus, Lactobacillus acidophilus,Lactobacillus brevis, Lactobacillus salivarius ssp salivarius,Lactobacillus farciminis, Lactococcus lactis, Lactobacillus casei,Lactobacillus salivarius, Lactobacillus rhamnosus, Clostridiumbutyricum, Aspergillus oryzae, Propionibacterium acidipropionici,Propionibacterium jensenii, Enterococcus faecium, Pediococcusacidilactici and combinations thereof.
 11. A method according to claim 1wherein the direct fed microbial is one or more of the followingstrains: Bacillus subtilis BS18 (NRRL B-50633), Bacillus subtilis BS278(NRRL 50634), Bacillus subtilis 4-7d (NRRL B-50505), Bacillus subtilis3-5h (NRRL B-50507), Bacillus subtilis AGTP BS3BP5 (NRRL B-50510),Bacillus subtilis BS918 (NRRL B-50508), Bacillus subtilis AGTP BS1013(NRRL-50509), B. subtilis AGTP 944 (NRRL B-50548), Bacillus subtilisAGTP BS442 (NRRL B-50542), B. subtilis AGTP BS1069 (NRRL B-50544), B.subtilis AGTP BS521 (NRRL B-50545), B. subtilis BS2084 (NRRL B-50013),B. subtilis LSSA01 (NRRL B-50104), B. subtilis B27 (NRRL B-50105), B.subtilis 3A-P4 (PTA-6506), Bacillus subtilis 15A-P4 (PTA-6507), B.subtilis 22C-P1 (PTA-6508), B. subtilis BL21 (NRRL B-50134), Bacilluslicheniformis BL21 (NRRL B-50134), Bacillus licheniformis 3-12a (NRRLB-50504), Bacillus licheniformis 4-2a (NRRL B-50506), Bacilluslicheniformis 842 (NRRL B-50516), Propionibacterium acidipropionici P261(NRRL B-50131), Propionibacterium acidipropionici P179 (NRRL B-50133),Propionibacterium acidipropionici P169 (PTA 5271), Propionibacteriumacidipropionici P170 (PTA 5272), Propionibacterium jensenii P63 (NRRLB-30979), Propionibacterium jensenii P195 (NRRL B-50132), Lactococcuslactis ID7 (PTA 6103), Lactococcus lactis JD19 (PTA 6104), Lactobacillusacidophilus A2020 (NRRL B-30977), Lactobacillus acidophilus A4000h (NRRLB-30978), Lactobacillus acidophilus PIBc6 (NRRL B-50103), Lactobacillusbrevis LBR 1000 (NRRL B-30982), Lactobacillus casei LC222 (NRRLB-30983), Lactobacillus johnsonii PLCB6 (NRRL B-50518), Lactobacillussalivarius o246i33w (NRRL B-50102), Lactobacillus brevis AJ25(PTA-6099), Lactobacillus brevis HE17 (PTA-6100), Lactobacillus brevis1E-1 (PTA-6509), Lactobacillus lactis C115 (PTA-6101), Lactobacilluslactis DJ6 (PTA-6102), Lactobacillus rhamnosus (CNCM 1-3698),Lactobacillus farciminis (CNCM 1-3699), Enterococcus faecium EF141(EN-1) (NRRL B-30981), Enterococcus faecium 2-id (NRRL B-50519),Pediococcus acidilactici PIJe3 (NRRL B-50101), Pediococcus acidilacticio246e42 (NRRL B-50171) and combinations thereof.
 12. A method accordingto claim 1 wherein the phytase is a 6-phytase or a 3-phytase.
 13. Amethod according to claim 12 wherein the phytase is a 6-phytase.
 14. Amethod according to claim 1 wherein the phytase is an E. coli phytase ora Buttiauxella phytase or a Citrobacter phytase or a Hafnia phytase oran Aspergillus phytase or a Penicillium phytase or a Trichoderma phytaseor an E. coli phytase or a Hansenula phytase or a Peniphora phytase. 15.A method according to claim 1 wherein the DFM is present at a dosage of3.75×10⁷ CFU/g feed additive composition and 1×10¹¹ CFU/g feed additivecomposition.
 16. A method according to claim 1 wherein a clinical orsubclinical intestinal disease challenge is present in the subject. 17.A method according to claim 16 wherein the clinical or subclinicalintestinal disease challenge may be caused by a pathogenic bacteria,such as Clostridium perfringens or E.coli. 18.-30. (canceled)
 31. Amethod of preparing a feed additive composition, comprising admixing atleast one direct fed microbial with a phytase, such that the dosage ofphytase in the composition is: a. about 30,000 FTU/g composition or morewhen dosed in a feed at at least 50 g/metric ton (MT) of feed, b. about20,000 FTU/g composition or more when dosed in a feed at at least 75g/metric ton (MT) of feed, c. about 15,000 FTU/g composition or morewhen dosed in a feed at at least 100 g/metric ton (MT) of feed, d. about15,000 FTU/g composition or more when dosed in a feed at at least 100g/metric ton (MT) of feed, e. about 10,000 FTU/g composition or morewhen dosed in a feed at at least 150 g/metric ton (MT) of feed, f. about7,500 FTU/g composition or more when dosed in a feed at at least 200g/metric ton (MT) of feed, g. about 5,000 FTU/g composition or more whendosed in a feed at at least 300 g/metric ton (MT) of feed, and thedosage of the direct fed microbial in the feed additive composition in arange from 2.5×10³ CFU DFM: 1 FTU enzyme to 6.7×10⁶ CFU:1 FTU enzyme,and optionally packaging.
 32. A method according to claim 31 wherein thedosage of the direct fed microbial in the feed additive composition in arange from 3.8×10³ CFU DFM: 1 FTU enzyme to 2.0×10⁵ CFU:1 FTU enzyme.33.-40. (canceled)